Subtilase Variants Having Altered Immunogenicity

ABSTRACT

The present invention relates to subtilase subtilases with an altered immunogenicity, particularly subtilases with a reduced allergenicity. Furthermore, the invention relates to expression of said subtilase variants and subtilases and to their use, such as in detergents and oral care products.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/547,555 filed on Oct. 24, 2006 (pending), which is a 35 U.S.C. 371national application of PCT/DK2005/000225 filed Apr. 1, 2005, whichclaims priority or the benefit under 35 U.S.C. 119 of Danish applicationno. PA 2004 00535 filed Apr. 2, 2004 and U.S. provisional applicationNo. 60/564,131 filed Apr. 21, 2004, the contents of which are fullyincorporated herein by reference.

FIELD OF INVENTION

The present invention relates to subtilase variants having alteredimmunogenicity, to the use thereof, as well as to a method for producingsaid subtilases and subtilase variants.

BACKGROUND OF THE INVENTION

An increasing number of proteins, including enzymes, are being producedindustrially, for use in various industries, housekeeping and medicine.Being proteins they are likely to stimulate an immunological response inman and animals, e.g. an allergic response.

Various attempts to alter the immunogenicity of proteins have beenconducted. In general it is only localized parts of the protein, knownas epitopes, which are responsible for induction of an immunologicresponse. An epitope consist of a number of amino acids, which may inthe primary sequence be sequential but which more often are located inproximity of each other in the 3-dimensional structure of the protein.It has been found that small changes in an epitope may affect thebinding to an antibody. This may result in a reduced importance of suchan epitope, maybe converting it from a high affinity to a low affinityepitope, or maybe even result in epitope loss, i.e. that the epitopecannot sufficiently bind an antibody to elicit an immunogenic response.

Another method for altering the immunogenicity of a protein is bymasking the epitopes by e.g. adding compounds, such as PEG, to theprotein.

WO 00/26230 and WO 01/83559 disclose two different methods of selectinga protein variant having reduced immunogenicity as compared to theparent protein.

WO 99/38978 discloses a method for modifying allergens to be lessallergenic by modifying the IgE binding sites.

WO 99/53038 discloses mutant proteins having lower allergenic responsein humans and methods for constructing, identifying and producing suchproteins.

Subtilases, which have a wide-spread use within the detergent industry,is a group of enzymes which potentially may elicit an immunogenicresponse, such as allergy. Thus there is a constant need for subtilasesor subtilase variants which have an altered immunogenicity, particularlya reduced allergenicity and which at the same still maintain theenzymatic activity necessary for their application.

WO 00/22103 discloses polypeptides with reduced immune response and WO01/83559 discloses protein variants having modified immunogenicity.

BRIEF DESCRIPTION OF THE INVENTION

In a first aspect, the present invention relates to variants of theparent subtilase enzyme of SEQ ID NO. 1, or a subtilase having at least80% homology, preferably 85%, or 90%, or 95%, or 98%, or 99% homology tothe parent enzyme, the variants having altered immunogenicity ascompared to the parent subtilase enzyme. The amino acid sequence of thevariants differ from the amino acid sequence of the parent subtilaseenzyme with respect to at least two mutations, at least one of themutations occurring in one of four identified epitopes of the parentsubtilase enzyme.

In a second aspect, the present invention relates to a variant of thesubtilase of SEQ ID NO. 1, wherein Glutamic acid has been inserted afterposition 99 (*99aE).

In a third aspect, the present invention relates to variants of SEQ IDNO. 1, wherein two or more mutations occur in the identified epitopes.The said mutations may occur in combination with the insertion ofGlutamic acid after position 99 (*99aE).

In a fourth aspect, the present invention relates to variants of SEQ IDNO.1 wherein mutations outside the epitopes occurs at one or more of thepositions 22, 141, 191, 247, 252, and 259. The said mutations may occurin combination with the insertion of Glutamic acid after position 99(*99aE).

In a fifth aspect, the present invention relates variants from the groupconsisting of S57P+*99aE+R247Q, and *99aE+A158V, and *99aE+E136G, and*99aE+E136K, and I79T+*99aE+Q191E, and *99aE+S141N+S156D, andT58A+*99aE+S156N, and *99aE+S141G+S156N+Q191E, andS78P+*99aE+N185S+Q206L, and *99aE+G195P+T260L, and*99aE+G160S+G211S+T260N, and *99aE+G195D+G211N+T260L, and*99aE+G160S+G195P+G211S+T260N, and *99aE+A158N+S161D, and*99aE+S259N+T260I, and *99aE+S259N, and *99aE+S259R, andT22A+*99aE+G160N+T260L, and *99aE+G160D+G195P+G211P+T260N, and*99aE+G160S+G211D+T260L.

In a sixth aspect, the present invention relates to variants of SEQ IDNO.1 with reduced immunogenicity as measured by epithelia assay.

In a seventh aspect, the present invention relates to variants of SEQ IDNO.1 with reduced immunogenicity as measured by MINT studies or byC-ELISA.

In still further aspects, the present invention relates to a DNAsequence encoding a subtilase and/or a subtilase variant of the presentinvention and to a vector comprising said DNA sequence, and to a hostcell comprising said vector.

In a final aspect, the present invention relates to a compositioncomprising a subtilase and/or a subtilase variant of the presentinvention.

Definitions

The term “subtilase” is in the context of the present invention to beunderstood as a sub-group of serine proteases as described by Siezen etal., Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6(1997) 501-523.

pIg is shorthand for a mixture of poly Immunoglobulins.

The term “parent” is in the context of the present invention to beunderstood as a protein, which is modified to create a protein variant.The parent protein may be a naturally occurring (wild-type) polypeptideor it may be a variant thereof prepared by any suitable means. Forinstance, the parent protein may be a variant of a naturally occurringprotein which has been modified by substitution, chemical modification,deletion or truncation of one or more amino acid residues, or byaddition or insertion of one or more amino acid residues to the aminoacid sequence, of a naturally-occurring polypeptide. Thus the term“parent subtilase” refers to a subtilase which is modified to create asubtilase variant.

The term “variant” is in the context of the present invention to beunderstood as a protein which has been modified as compared to a parentprotein at one or more amino acid residues.

The term “mutation(s)”, “modification(s)” or “modified” is in thecontext of the present invention to be understood as to include chemicalmodification of a protein as well as genetic manipulation of the DNAencoding a protein. The modification(s) may be replacement(s) of theamino acid side chain(s), substitution(s), deletion(s) and/or insertionsin or at the amino acid(s) of interest. Thus the term “modifiedprotein”, e.g. “modified subtilase”, is to be understood as a proteinwhich contains modification(s) compared to a parent protein.

The term “position” is in the present invention to be understood as thenumber from the N-terminal end of an amino acid in a protein. Theposition numbers used in the present invention refer to the positions ofSubtilisin Novo (BPN′) (SEQ ID NO: 5) from B. amyloliquefaciens.However, other subtilases are also covered by the present invention. Thecorresponding positions of other subtilases are defined by alignmentwith Subtilisin Novo (BPN′) (SEQ ID NO: 5) from B. amyloliquefaciens byusing the GAP program. GAP is provided in the GCG program package(Program Manual for the Wisconsin Package, Version 8, August 1994,Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711)(Needleman, S. B. and Wunsch, C. D., (1970), Journal of MolecularBiology, 48, 443-45). Unless specified, positions mentioned in thepresent invention, are given in the BPN′ numeration, and can beconverted by alignment.

The term “protein” is in the context of the present invention intendedto cover oligopeptides, polypeptides as well as proteins as such.

The term “deletion” or “deleted”, used in relation to a position or anamino acid, refers in the context of the present invention to that theamino acid in the particular position has been deleted or that it isabsent.

The term “insertion” or “inserted”, used in relation to a position oramino acid, refers in the context of the present invention to that 1 ormore amino acids, e.g. between 1-5 amino acids, have been inserted orthat 1 or more amino acids, e.g. between 1-5 amino acids are presentafter the amino acid in the particular position

The term “substitution” or “substituted”, used in relation to a positionor amino acid, refers in the context of the present invention to thatthe amino acid in the particular position has been replaced by anotheramino acid or that an amino acid different from the one of a specifiedprotein, e.g. protein sequence, is present.

Amino Acids

The well-known three-letter and one-letter abbreviations for amino acidsis used (see e.g. Creighton T E (1993), Proteins; Structures andMolecular Properties, 2^(nd) Edition W.H: Freeman and Company, FIG. 1.1,p. 3). The abbreviation “X” or “Xaa” is used for any amino acid. Withinthe context of the present invention the abbreviation “aa” is used for“amino acid”.

Variants

To describe a deletion, an insertions and/or a substitution of aminoacid(s) the following nomenclature is used in the present invention.

Original amino acid(s), position(s), deleted/inserted/substituted aminoacid(s)

According to this the substitution of Glutamic acid for glycine inposition 195 is designated as:

-   -   Gly 195 Glu or G195E

a deletion of glycine in the same position is:

-   -   Gly 195* or G195*

Where a deletion in comparison with the sequence used for the numberingis indicated, an insertion in such a position is indicated as:

-   -   *36 Asp or *36D

for insertion of an aspartic acid in position 36.

Insertion of an additional amino acid residue such as Aspartic acid isindicated as:

-   -   *36a Asp or *36aD

for insertion of an aspartic acid just after position 36 in the parentprotein.

Multiple mutations are separated by pluses, i.e.:

-   -   Arg 170 Tyr+Gly 195 Glu or R170Y+G195E

representing mutations in positions 170 and 195 substituting tyrosineand glutamic acid for arginine and glycine, respectively.

Multiple mutations may also be separated by commas, i.e.:

-   -   Arg 170 Tyr, Gly 195 Glu or R170Y,G195E

representing mutations in positions 170 and 195 substituting tyrosineand glutamic acid for arginine and glycine, respectively.

The “immunogenicity” of a compound used in connection with the presentinvention refers to the ability of this compound to induce an‘immunological response’ in animals including man. The term“immunological response”, used in connection with the present invention,is the response of an organism to a compound, which involves the immunesystem according to any of the four standard reactions (Type I, II, Illand IV according to Coombs & Gell).

The immunogenicity can be assessed by animal experiments (e.g. MINT, andSC-mouse); C-ELISA (competitive ELISA) and epithelia cell assay areother predictors of immunogenicity

The “allergenicity” of a compound used in connection with the presentinvention refers to the ability of this compound to induce an ‘allergicresponse’ in animals including man.

The term “allergic response”, used in connection with the presentinvention, is the response of an organism to a compound, which involvesIgE mediated responses (Type I reaction according to Coombs & Gell). Itis to be understood that sensitisation (i.e. development ofcompound-specific IgE antibodies) upon exposure to the compound isincluded in the definition of “allergic response”.

“Homology” or “homologous to” is in the context of the present inventionto be understood in its conventional meaning and number the “homology”between two amino acid sequences should be determined by use of the“Similarity” defined by the GAP program from the University of WisconsinGenetics Computer Group (GCG) package using default settings foralignment parameters, comparison matrix, gap and gap extensionpenalties. Default values for GAP penalties, i.e. GAP creation penaltyof 3.0 and GAP extension penalty of 0.1 (Program Manual for theWisconsin Package, Version 8, August 1994, Genetics Computer Group, 575Science Drive, Madison, Wis., USA 53711). The method is also describedin S. B. Needleman and C. D. Wunsch, Journal of Molecular Biology, 48,443-445 (1970). Identities can be extracted from the same calculation.The homology between two amino acid sequences can also be determined by“identity” or “similarity” using the GAP routine of the GCG packageversion 9.1 with default setting for alignment parameters, comparisonmatrix, gap and gap extension penalties can also be applied onsubtilases using the following parameters: gap creation penalty=8 andgap extension penalty=8 and all other parameters kept at their defaultvalues. The output from the routine is besides the amino acid alignmentthe calculation of the “Percent Identity” and the “Similarity” betweenthe two sequences. The numbers calculated using GCG package version 9.1is slightly different from the version 8.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the specific IgE-response at day 31 following IntranasalInstillation of the subtilase of SEQ ID NO 1 and two of the subtilasesof the present invention (5P and 10P) into BDF1 mice, group mean values.

FIGS. 2 a-c show individual values of the specific IgE-responses at day31 following Intranasal Instillation of the subtilase of SEQ ID NO 1 andof two of the subtilases of the present invention (5P and 10P) into BDF1mice.

FIG. 3 shows a M-CSF-enzyme dose/response curve for human epithelialcell cultures exposed to the enzymes of SEQ ID NO1 and SEQ ID NO1 with a*99aE insertion.

FIG. 4 shows an alignment between subtilisin BPN′ (a) and Savinase (SEQID NO 1). The alignment can be obtained by the GAP routine of the GCGpackage version 9.1 to number the variants using the followingparameters: gap creation penalty=8 and gap extension penalty=8 and allother parameters kept at their default values.

SEQUENCE LISTING

The present application contains information in the form of a sequencelisting, which is appended to the application and also submitted on adata carrier accompanying this application. The contents of the datacarrier are fully incorporated herein by reference.

DETAILED DESCRIPTION OF THE INVENTION Subtilase Variants of theInvention

The present invention relates to subtilase variants of SEQ ID NO 1(Savinase), wherein two or more mutations have been introduced, with atleast one of the mutations in one of the four epitopes of the parentsubtilase. The inventors have found that said subtilase variants have analtered immunogenicity in comparison to the parent subtilase (Savinase).

The amino acids in positions of a subtilase variant of the presentinvention may be modified by genetic manipulation of the DNA encodingthe parent subtilase. In particular said positions may be modified bygenetic manipulation of the DNA encoding the parent subtilase, e.g. bydeletion, insertion or substitution. A mutation may typically involvemodification of between 1 to 5 amino acids, such as 1, 2, 3, 4 or 5amino acids.

Mutations may occur in between 1 to 4 epitopes, such as 1, 2, 3, or 4epitopes.

In a particular embodiment of the invention, one of the mutations is theinsertion of Glutamic acid (E) after position 99 (mutation stated as*99aE). Further mutations outside the epitopes may occur at one or moreof the positions 22 (for example substitution with Alanine (T22A)), 141(for example substitution with Asparagine (S141N) or Glycine (S141G)),191 (for example substitution with Glutamic acid (Q191E)), and 247 (forexample substitution with Glutamine (R247Q)).

In another particular embodiment of the invention, one may createlibraries of genes that have been modified in one or more of theepitopes in a stochastic manner, e.g. such that several of the aminoacids occurring in epitopes have been diversified by doped oligomermutagenesis. Such libraries can then be screened for active variants(e.g. by plating transformed Bacillus colonies on skim-milk and growthmedia-containing agar plates) and the clones that express an activevariant selected for sequencing of the relevant variant gene.

Mutations in the epitopes may occur at one or more of the positions 57,58, and 136, wherein when a substitution occurs at position 57 thesubstituting amino acid may be Proline; when a substitution occurs atposition 58 the substituting amino acid may be Alanine, and when asubstitution occurs at position 136 the substituting amino acid may beselected from the group consisting of Glycine, and Lysine; or mutationsin the epitopes may occur at one or more of the positions 156, 158, 160,161, and 195, wherein when a substitution occurs at position 156 thesubstituting amino acid may be selected from the group consisting ofAspartic acid, and Asparagine; when a substitution occurs at position158 the substituting amino acid may be selected from the groupconsisting of Valine, and Asparagine; when a substitution occurs atposition 160 the substituting amino acid may be selected from the groupconsisting of Serine, Asparagine, and Aspartic Acid; when a substitutionoccurs at position 161 the substituting amino acid may be Aspartic Acid;when a substitution occurs at position 195 the substituting amino acidmay be Glutamic acid; or mutations in the epitopes may occur at one ormore of the positions 78, 79, 206, and 211, wherein when a substitutionoccurs at position 78 the substituting amino acid may be Proline; when asubstitution occurs at position 79 the substituting amino acid may beThreonine; when a substitution occurs at position 206 the substitutingamino acid may be Leucine; when a substitution occurs at position 211the substituting amino acid may be selected from the group consisting ofSerine, Proline, and Aspartic acid; or mutations in the epitopes mayoccur at one or more of the positions 158, 160, 161, 185, 195, 259, and260, wherein when a substitution occurs at position 158 the substitutingamino acid may be selected from the group consisting of Valine, andAsparagine; when a substitution occurs at position 160 the substitutingamino acid may be selected from the group consisting of Serine,Asparagine, and Aspartic Acid; when a substitution occurs at position161 the substituting amino acid may be Aspartic Acid; when asubstitution occurs at position 185 the substituting amino acid may beSerine; when a substitution occurs at position 195 the substitutingamino acid may be Glutamic acid; when a substitution occurs at position259 the substituting amino acid may be selected from the groupconsisting of Asparagine, and Arginine; when a substitution occurs atposition 260 the substituting amino acid may be selected from the groupconsisting of Leucine, Isoleucine, and Asparagine.

In particular the subtilase variant of the present invention may be oneof the following: I79T+*99aE+Q191E; and *99aE+G195P+T260L; and*99aE+G160D+G195P+G211P+T260N.

In another particular embodiment, the present invention relates tovariants with improved wash performance.

In a further embodiment, the present invention relates to expression ofsubtilase variants of the present invention.

In yet another embodiment, the present invention relates tocompositions, in particular cleaning and personal care compositions,comprising a subtilase variant of the present invention.

Subtilase

Subtilases constitute a sub-group of serine protease according to Siezenet al., Protein Engng. 4 (1991) 719-737 and Siezen et al. ProteinScience 6 (1997) 501-523. Subtilases are defined by homology analysis ofmore than 170 amino acid sequences of serine proteases previouslyreferred to as subtilisin-like proteases. The subtilases may be dividedinto 6 sub-divisions, i.e. the Subtilisin family, the Thermitase family,the Proteinase K family, the Lantibiotic peptidase family, the Kexinfamily and the Pyrolysin family. The Subtilisin family may be furtherdivided into 3 sub-groups, i.e. I-S1 (“true” subtilisins), I-S2 (highlyalkaline proteases) and intra-cellular subtilisins. Definitions orgrouping of enzymes may vary or change, however, in the context of thepresent invention the above division of subtilases into sub-division orsub-groups shall be understood as those described by Siezen et al.,Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6(1997) 501-523.

The subtilase variants of the present invention are obtained bymodification of the parent subtilase of SEQ ID NO. 1 or a subtilasehaving at least 80% homology, preferably 85%, or 90%, or 95%, or 98%, or99% homology to the parent subtilase of SEQ ID NO. 1.

The parent subtilase and/or the subtilase of the present invention maybe a subtilase isolated from natural source, i.e. a wild type subtilase,or it may be a subtilase isolated from a natural source in whichsubsequent modifications have been made while retaining thecharacteristic of a subtilase. Examples of such subtilase variants whichmay be parent subtilases include those disclosed in EP 130.756, EP214.435, WO 87/04461, WO 87/05050, EP 251.446, EP 260.105, WO 88/08028,WO 88/08033, WO 89/06279, WO 91/00345, EP 525 610 and WO 94/02618. Inanother embodiment the parent subtilase may be a subtilase which hasbeen prepared by a DNA shuffling technique, such as described by J. E.Ness et al., Nature Biotechnology, 17, 893-896 (1999). Further, a parentsubtilase may be constructed by standard techniques for artificialcreation of diversity, such as by DNA shuffling of different subtilasegenes (WO 95/22625; Stemmer W P C, Nature 370:389-91 (1994)).

The activity of subtilases and subtilase variants can be determined asdescribed in “Methods of Enzymatic Analysis”, third edition, 1984,Verlag Chemie, Weinheim, vol. 5.

In Silico Identification of Epitope Patterns and Epitopes in SubtilaseEnzymes.

Subtilase enzymes may be epitope mapped using the proprietary in silicoepitope mapping tool disclosed in detail in WO 00/26230 and WO 01/83559.In brief, this tool comprises a database of epitope patterns (determinedfrom an input of peptide sequences, known to bind specifically toanti-protein antibodies) and an algorithm to analyse 3-D structure of agiven protein against the epitope pattern database. This will determinethe possible epitopes on that protein, and the preference of each aminoacid in the protein sequence to be part of epitopes.

The term “epitope pattern” as used herein is to be understood as aconsensus sequence of antibody binding peptides. An example is theepitope pattern A R R * R. The sign “*” in this notation indicates thatthe aligned antibody binding peptides included a non-consensus moietybetween the second and the third arginine. That moiety may be any aminoacid or a few amino acids or no amino acid. Epitope patterns are used toidentify epitopes and minimal epitopes on complex antigens.

Identifying Antibody-Binding Peptides

Antibody-binding peptides can be identified by many different ways. Oneis to synthesize a number of peptides of known sequence, and test fortheir ability to bind antibodies of interest, e.g., in ELISA or otherimmunochemical assays. Such data are available in great abundance in theliterature.

A particularly effective way is to prepare a library of many differentrandom peptide sequences and select experimentally only the ones thatbind antibodies well and specific (i.e., can compete out the proteintowards which the antibodies were raised). Phage display techniques arewell suited for this way of finding antibody binding peptides:

In a phage display system, a sequence encoding a desired amino acidsequence is incorporated into a phage gene coding for a proteindisplayed on the surface of the phage. Thus, the phage will make anddisplay the hybrid protein on its surface, where it can interact withspecific target agents. Given that each phage contains codons for onespecific sequence of a determined length, an average phage displaylibrary can express 10⁸-10¹² different random sequences. If thedisplayed sequence resembles an epitope, the phage can be selected by anepitope-specific antibody. Thus, it is possible to select specificphages from the bulk of a large number of phages, each expressing theirone hybrid protein.

It is important that the amino acid sequence of the (oligo)peptidespresented by the phage display system have a sufficient length topresent a significant part of an epitope to be identified. Theoligopeptides may have from 5 to 25 amino acids, preferably at least 8amino acids, such as 9 amino acids.

The antibodies used for reacting with the oligopeptides can bepolyclonal or monoclonal. In particular, they may be IgE antibodies toensure that the epitopes identified are IgE epitopes, i.e., epitopesinducing and binding IgE. The antibodies may also be monospecific,meaning they are isolated according to their specificity for a certainprotein. Polyclonal antibodies are preferred for building up data onantibody-binding peptides to be used in the in silico mapping tool inorder to obtain a broader knowledge about the epitopes of a polypeptide.

These reactive peptides, by virtue of their reactivity againstantibodies, to some degree resemble the appearance of an epitope on afull polypeptide.

Identifying Epitope Patterns from Reactive Peptides

The reactive (oligo)peptides identified e.g. by phage display arecompared and aligned in order to identify common epitope patterns, whichthen can be used for identification of antibody binding epitopes on a3-dimensional polypeptide.

In the alignment conservative alternatives to an amino acid such asAspartate and Glutamate, Lysine and Arginine, Serine and Threonine areconsidered as one or equal. Thus, the alignment results in a number ofpatterns, which depend on the chosen number of residues of the peptides.Using for example a 7-mer peptide, the pattern may have the form:

X X * * X X X,

where “*” in this notation indicates a non-consensus moiety which may beany amino acid or group of amino acids or no amino acid, while X is oneof the following 13 residue types: AG, C, DE, FY, H, IL, KR, M, NQ, P,ST, V, and W, where the pairs AG, DE, FY, IL, KR, NQ, ST areconservative alternatives and considered equal. Accordingly, 3 peptidessuch as

A K S N N K R (SEQ ID NO: 2) A K S M N K R (SEQ ID NO: 3) A K T P N K K(SEQ ID NO: 4)would create a pattern of [AG] [KR] [ST] * [NQ] [KR] [KR], where theresidues AG KR ST and NQ KR KR are consensus residues shared by all 3peptides and thus the epitope pattern would be AG KR ST * NQ KR KR. Thepatterns are chosen to describe a complete set of reactive(oligo)peptides (obtained e.g., by a phage display and antibodyreaction) by the fewest possible patterns.

The epitope patterns may be determined directly from the reactivepeptides; if for example a library of 7-mer reactive peptides is made,one can use each different reactive 7mer peptide, taking conservativealternatives into account, as an epitope pattern in the epitope mappingapproach as described below.

It is also possible to reduce the number of epitope patterns to beexamined in the epitope mapping by removing redundant patterns and/or byemploying experimental designs as known in the art (See example 1).

Epitope Mapping Algorithm

When epitope patterns have been identified they are subsequentlycompared to the three-dimensional coordinates of the amino acid sequenceof the polypeptide of interest, in order to identify combinations ofresidues on the polypeptide surface corresponding to the consensussequence(s) or epitope pattern(s). In this way, amino acids residues,which are important for antibody binding, can be identified.

Once one or more epitope patterns have been identified, any polypeptidefor which a three-dimensional structure is known may be analysed forepitopes matching the epitope patterns. Finding an epitope on apolypeptide is achieved by searching the surface of the polypeptide inthe following way:

-   (1) For all amino acids in the polypeptide it is examined if (a) the    amino acid type match the first amino acid of an epitope pattern    and (b) the surface accessibility greater than or equal to a chosen    threshold allowing the amino acid to be immunological interactive.    Those amino acid satisfying 1(a) and 1(b) are selected.-   (2) For all amino acids within a selected distance (e.g. 10    Angstroms) of the amino acids selected in step 1 it is examined    if (a) the amino acid type matches the second amino acid of the    pattern and (b) the surface accessibility greater than or equal to a    chosen threshold allowing the amino acid to be immunological    interactive. Those amino acid satisfying 2(a) and 2(b) are selected-   (3) For all amino acids within a selected distance (e.g. 10    Angstroms) of the amino acids selected in step 2 it is examined    if (a) the amino acid type matches the third amino acid of the    pattern and (b) the surface accessibility greater than or equal to a    chosen threshold allowing the amino acid to be immunological    interactive. Those amino acid satisfying 3(a) and 3(b) are selected.

This procedure (step 3) is repeated for all amino acids in the epitopepattern consensus sequence. The coordinates of its C-alpha atom definethe spatial positioning of an amino acid. The surface solventaccessibility threshold is given in percent of an average for theparticular residue type (see example 1).

If matching amino acids for all amino acids in the epitope pattern canbe found in the structure of the polypeptide it is a very strongindication that an epitope has been found.

However it is also checked that the size of the epitope is satisfactory,i.e., the distance between any two residues is below a given threshold,usually 25 Å.

The epitopes found may be ranked and weighted according to their totalaccessible surface area, in order to improve further the predictabilityof the tool.

Finally, when all possible epitopes have been mapped for the protein ofinterest, one can provide a score for each amino acid of the protein byadding up the number of times it appears in an epitope pattern. Thisscore will be an indication of the likelihood that modification(substitution, insertion, deletion, glycosylation or chemicalconjugation) of that amino acid will, result in a variant with a lowerantigenicity. All amino acids of the protein can then be rankedaccording to this score and those with highest scores can be selectedfor mutagenesis.

The epitope mapping tool can be adjusted, such that only a subset of theknown reactive peptides are included as data set for building epitopepatterns, and thus for conducting epitope mapping. For instance, one maychoose only to include peptides reactive to IgE antibodies (rather thanto IgG or other antibodies), or one may include only peptides reactiveto human antibodies etc. One may choose to involve only peptidesreactive against the target protein in order to get a more specificresponse, however, in general, peptides reactive to antibodies that inturn were raised against any protein are included.

If no three-dimensional structure coordinates are available for theprotein of interest, one can map the epitope patterns directly to theprimary sequence of the protein of interest.

From all the above information, it is obvious, that the epitopes areconveniently determined using this epitope mapping tool.

Further, the in silico epitope mapping tool can be used to predict ifmutating one amino acid residue will result in that the new variantoverall will have fewer epitopes. Thus, some or all 19 possiblesubstitutions can be tested in a given position, the epitope mappingprocedure repeated for a model structure of each of these proposedvariants, and the best variant(s) can be constructed by mutation andtested experimentally.

Identified Epitopes of Parent Subtilase

Using the epitope mapping tool the present inventors have identifiedfour epitopes corresponding to the following positions of SEQ ID NO 1:

-   Epitope 1: P52-N62; A98-S105; G127-E136; Y167; R170.-   Epitope 2: G127; P129; N155-S161; Y167; R170; S188; Y192-G195; N218;    N261-L262.-   Epitope 3: A1-W6; P14-N18; T38-I44; Q59-N62; L75-I79; S87; N155;    N204-N218.-   Epitope 4: N6; Q12; G157-S161; D181-N185; Y192-G195; T255-S265.

Selection of Positions for Mutation

Once the epitopes of a polypeptide have been determined, variants of thepolypeptide with modified antigenic properties can be made by mutatingone or more of the amino acid residues comprised in the epitope. In thiscontext mutation encompasses deletion and/or substitution of an aminoacid residue and/or insertion of one or more amino acids before or afterthat residue.

When providing a polypeptide variant suitable as an enzyme, it isparticularly desirable to alter IgE epitopes to reduce the binding ofIgE, while at the same time maintaining the activity and stability ofthe variant, hence it is desirable that the polypeptides retains thethree-dimensional conformation of the parent polypeptide.

The epitope identified may be mutated by substituting at least one aminoacid of the epitope. The mutation will often be a substitution with anamino acid of different size, hydrophilicity, polarity and/or acidity,such as a small amino acid in exchange of a large amino acid, ahydrophilic amino acid in exchange of a hydrophobic amino acid, a polaramino acid in exchange of a non-polar amino acid and a basic in exchangeof an acidic amino acid.

The mutation(s) performed may be performed by standard techniques wellknown to a person skilled in the art, such as site-directed mutagenesis(see, e.g., Sambrook et al. (1989), Molecular Cloning. A LaboratoryManual, Cold Spring Harbor, NY).

The mutagenesis may be spiked mutagenesis which is a form ofsite-directed mutagenesis, in which the primers used have beensynthesized using mixtures of oligonucleotides at one or more positions.

A general description of nucleotide substitution can be found in e.g.,Ford et al., 1991, Protein Expression and Purification 2, pp. 95-107.

The subtilisin variant of the present invention concerns variant whereat least one mutation is in one of the identified epitopes in order toalter the immunogenicity of the variant as compared to the parentsubtilase. Particularly the variant has altered antibody binding profileas compared the parent subtilase, more particularly combined withpreserved performance and/or activity and/or stability. Still morepreferably, the variant has an altered immunogenic profile in exposedanimals, including humans, as compared to the parent subtilase.

Further the variant induces an altered immunogenic response, preferablyreduced allergenic response, in exposed animals, including humans, ascompared to the parent subtilase.

Verification of Variants Having Altered Antigenic Properties

The mutation of amino acids, comprised in an epitope, will cause theantigenic properties of the polypeptide to change, as predicted by thein silico determination of the epitopes. However, the quantitativeeffect of the mutation on the antigenicity, i.e., the antibody binding,and the immunogenicity of the variant, is suitably determined usingvarious in vivo or in vitro model systems. The subtilase variants of thepresent invention may be tested for altered allergenicity and/orimmunogenicity by using a purified preparation of the subtilasevariants. Thus before testing the subtilase variants for alteredallergenicity and/or immunogenicity they may be expressed in largerscale and/or purified by conventional methods.

The antibody binding can be examined in detail using dose-responsecurves and e.g., direct or competitive ELISA (c-ELISA), such asdescribed in WO 99/47680, or by other or other solid phase immunoassaysor cellular assays.

In a particular embodiment the residual binding of the polypeptidevariant is at least 5%, such as 10%, or 20%, or 30%, more preferably atleast 40% such as 50%, or 60%, or 70%, most preferably at least 80%,such as 85%, or 90%, or 95%, or 98%, or 99%, or 100%.

In another particular embodiment the binding affinity is differentfrom 1. The binding affinity may be lower than 1 when compared to aparent enzyme, such as lower than 0.9, such as 0.8, or 0.7, or 0.6, orlower than 0.5, such as 0.4, or 0.3, or 0.2, or lower than 0.15, such as0.1, or 0.05, or 0.01. The binding affinity may also be higher than 1when compared to a parent enzyme, such as higher than 1.5, or 2.0, or3.0, or 4.0, or 5.0, or higher than 7.0, such as 10, or 15, or 20, or30, or higher than 40, such as 50, or 60, or 70, or 80, or 90, or 100,or 150, or 200.

In a particular embodiment in vitro screening for reduced allergenicityand adjuvancy can be performed on a human airlifted epithelial cellculture assay. Human lung epithelial cells are seeded on polycarbonatetissue culture insert, placed in wells on a tissue culture plate. Thecells are cultured until confluent. Then the medium in the insert isremoved and the cells are cultured for 3 more days at the liquid-airinterface thus created. The cells are then stimulated with increasingdoses of enzyme for 4 hours. Then supernatants are harvested and M-CSF(Macrophage colony-stimulating factor) release triggered to enzyme inthe supernatant is measured. Variants with reduced allergenicity andadjuvancy require higher doses in order to elicit comparable M-CSFresponses with parent protein. The epithelial assay as described inExample 7 shows that M-CSF production by epithelial cells in response tostimulation with enzyme variants requires higher enzyme doses forelicitation of M-CSF responses that are comparable with parent protein(table 3). In particular the M-CSF release triggered to enzyme variantsas compared with parent protein is reduced at least 2 times, preferablyat least 5, 10, 25 times, or 40, 60, 80, 100 times, or even 150 or 200times for enzyme variants with a reduced allergenic potency. Inaddition, the toxic potency of the variants determined by a fall inM-CSF release by epithelial cells when triggered by higher amounts ofprotease is reduced.

In a particular embodiment the in vivo verification comprises skin pricktesting (SPT), in which a subtilase allergic subject/individual isexposed to Subtilase on the skin, followed by puncture with a needle,where after the IgE reactivity is measured as the diameter of the whealand flare reaction, in response to a polypeptide variant of theinvention is compared to that to the parent subtilase (Kronquist et al.,Clin. Exp. Allergy, 2000, vol. 30, pp. 670-676).

The in vivo immunogenic properties of the polypeptide variant of theinvention may also suitably be measured in an animal test, wherein testanimals are exposed to a parent subtilase and the responses to variantsas well as to the parent subtilase allergen are measured. The immuneresponse measurements may include comparing reactivity of serum IgE orT-cells from a test animal with a parent subtilase and the subtilasevariant.

In a particular embodiment the in vivo verification comprises exposing amouse to a parent subtilase by the intranasal route, and verifying thatserum IgE is less reactive with a subtilase variant than with the parentsubtilase. Useful in vivo animal models include the mouse intranasaltest (MINT) model (Robinson et al., Fund. Appl. Toxicol. 34, pp. 15-24,1996). Thus the term reduced allergenicity used in connection with thesubtilases variants/subtilases of the present invention is to beunderstood as an IgE response which is less or none in said assaycompared to the parent subtilase. In particular the IgE level measuredin said assay obtained in response to said subtilase variants and/orsubtilases may be 35%, such as 30% or 25% or 20% or 15% or 10% of theIgE level obtained in response to the parent subtilase/Savinase,respectively.

Further the in vivo verification may comprise exposing a test animal toa polypeptide variant by the intratracheal route and verifying that thespecific IgE titres or IgG titres if in guinea pigs are lower than withthe parent subtilase. Useful in vivo animal models include the guineapig intratracheal (GPIT) model (Ritz, et al. Fund. Appl. Toxicol., 21,pp. 31-37, 1993) and the rat intratracheal (rat-IT) model (WO 96/17929,Novo Nordisk).

Still further the in vivo verification may comprise exposing a testanimal subcutaneously to the subtilase allergen and the polypeptidevariant. Also, IgE binding and cross reactivity can be measuredfollowing this route of exposure. A suitable model is the mousesubcutaneous (mouse-SC) model (WO 98/30682, Novo Nordisk).

Methods for Production of Subtilase Variants and Subtilases

The subtilase variants and subtilases of the present invention may beproduced by any known method within the art and the present inventionalso relates to nucleic acid encoding a subtilase variant or subtilaseof the present invention, a DNA construct comprising said nucleic acidand a host cell comprising said nuclei acid sequence.

In general natural occurring proteins may be produced by culturing theorganism expressing the protein and subsequently purifying the proteinor it may be produced by cloning a nucleic acid, e.g. genomic DNA orcDNA, encoding the protein into an expression vector, introducing saidexpression vector into a host cell, culturing the host cell andpurifying the expressed protein.

Typically protein variants may be produced by site-directed mutagenesisof a parent protein, introduction into expression vector, host cell etc.The parent protein may be cloned from a strain producing the polypeptideor from an expression library, i.e. it may be isolated from genomic DNAor prepared from cDNA, or a combination thereof.

In general standard procedures for cloning of genes and/or introducingmutations (random and/or site directed) into said genes may be used inorder to obtain a parent subtilase, or subtilase or subtilase variant ofthe invention. For further description of suitable techniques referenceis made to Molecular cloning: A laboratory manual (Sambrook et al.(1989), Cold Spring Harbor lab., Cold Spring Harbor, N.Y.; Ausubel, F.M. et al. (eds.)); Current protocols in Molecular Biology (John Wileyand Sons, 1995; Harwood, C. R., and Cutting, S. M. (eds.)); MolecularBiological Methods for Bacillus (John Wiley and Sons, 1990); DNACloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985);Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds (1985)); TranscriptionAnd Translation (B. D. Hames & S. J. Higgins, eds. (1984)); Animal CellCulture (R. I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRLPress, (1986)); A Practical Guide To Molecular Cloning (B. Perbal,(1984)) and WO 96/34946.

Expression Vectors

A recombinant expression vector comprising a nucleic acid sequenceencoding a subtilase or subtilase variant of the invention may be anyvector that may conveniently be subjected to recombinant DNA proceduresand which may bring about the expression of the nucleic acid sequence.

The choice of vector will often depend on the host cell into which it isto be introduced. Examples of a suitable vector include a linear orclosed circular plasmid or a virus. The vector may be an autonomouslyreplicating vector, i.e., a vector which exists as an extra chromosomalentity, the replication of which is independent of chromosomalreplication, e.g., a plasmid, an extra chromosomal element, a minichromosome, or an artificial chromosome. The vector may contain anymeans for assuring self-replication. Examples of bacterial origins ofreplication are the origins of replication of plasmids pBR322, pUC19,pACYC177, pACYC184, pUB110, pE194, pTA1060, and pAMβ1. Examples oforigin of replications for use in a yeast host cell are the 2 micronorigin of replication, the combination of CEN6 and ARS4, and thecombination of CEN3 and ARS1. The origin of replication may be onehaving a mutation which makes it function as temperature-sensitive inthe host cell (see, e.g., Ehrlich, 1978, Proceedings of the NationalAcademy of Sciences USA 75:1433).

Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Vectors which areintegrated into the genome of the host cell may contain any nucleic acidsequence enabling integration into the genome, in particular it maycontain nucleic acid sequences facilitating integration into the genomeby homologous or non-homologous recombination. The vector system may bea single vector, e.g. plasmid or virus, or two or more vectors, e.g.plasmids or virus', which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon.

The vector may in particular be an expression vector in which the DNAsequence encoding the subtilase of the invention is operably linked toadditional segments or control sequences required for transcription ofthe DNA. The term, “operably linked” indicates that the segments arearranged so that they function in concert for their intended purposes,e.g. transcription initiates in a promoter and proceeds through the DNAsequence encoding the subtilase variant. Additional segments or controlsequences include a promoter, a leader, a polyadenylation sequence, apropeptide sequence, a signal sequence and a transcription terminator.At a minimum the control sequences include a promoter andtranscriptional and translational stop signals.

The promoter may be any DNA sequence that shows transcriptional activityin the host cell of choice and may be derived from genes encodingproteins either homologous or heterologous to the host cell.

Examples of suitable promoters for use in bacterial host cells includethe promoter of the Bacillus subtilis levansucrase gene (sacB), theBacillus stearothermophilus maltogenic amylase gene (amyM), the Bacilluslicheniformis alpha-amylase gene (amyL), the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), the Bacillus subtilis alkaline protease gene,or the Bacillus pumilus xylosidase gene, the Bacillus amyloliquefaciensBAN amylase gene, the Bacillus licheniformis penicillinase gene (penP),the Bacillus subtilis xylA and xylB genes, and the prokaryoticbeta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of theNational Academy of Sciences USA 75:3727-3731). Other examples includethe phage Lambda P_(R) or P_(L) promoters or the E. coli lac, trp or tacpromoters or the Streptomyces coelicolor agarase gene (dagA). Furtherpromoters are described in “Useful proteins from recombinant bacteria”in Scientific American, 1980, 242:74-94; and in Sambrook et al., 1989,supra.

Examples of suitable promoters for use in a filamentous fungal host cellare promoters obtained from the genes encoding Aspergillus oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, Fusariumoxysporum trypsin-like protease (as described in U.S. Pat. No.4,288,627, which is incorporated herein by reference), and hybridsthereof. Particularly preferred promoters for use in filamentous fungalhost cells are the TAKA amylase, NA2-tpi (a hybrid of the promoters fromthe genes encoding Aspergillus niger neutral (-amylase and Aspergillusoryzae triose phosphate isomerase), and glaA promoters. Further suitablepromoters for use in filamentous fungus host cells are the ADH3 promoter(McKnight et al., The EMBO J. 4 (1985), 2093-2099) or the tpiA promoter.

Examples of suitable promoters for use in yeast host cells includepromoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem.255 (1980), 12073-12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1(1982), 419-434) or alcohol dehydrogenase genes (Young et al., inGenetic Engineering of Microorganisms for Chemicals (Hollaender et al,eds.), Plenum Press, New York, 1982), or the TPI1 (U.S. Pat. No.4,599,311) or ADH2-4c (Russell et al., Nature 304 (1983), 652-654)promoters.

Further useful promoters are obtained from the Saccharomyces cerevisiaeenolase (ENO-1) gene, the Saccharomyces cerevisiae galactokinase gene(GAL1), the Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP),and the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene. Otheruseful promoters for yeast host cells are described by Romanos et al.,1992, Yeast 8:423-488. In a mammalian host cell, useful promotersinclude viral promoters such as those from Simian Virus 40 (SV40), Roussarcoma virus (RSV), adenovirus, and bovine papilloma virus (BPV).

Examples of suitable promoters for use in mammalian cells are the SV40promoter (Subramani et al., Mol. Cell Biol. 1 (1981), 854-864), the MT-1(metallothionein gene) promoter (Palmiter et al., Science 222 (1983),809-814) or the adenovirus 2 major late promoter. An example of asuitable promoter for use in insect cells is the polyhedrin promoter(U.S. Pat. No. 4,745,051; Vasuvedan et al., FEBS Lett. 311, (1992)7-11), the P10 promoter (J. M. Vlak et al., J. Gen. Virology 69, 1988,pp. 765-776), the Autographa californica polyhedrosis virus basicprotein promoter (EP 397 485), the baculovirus immediate early gene 1promoter (U.S. Pat. No. 5,155,037; U.S. Pat. No. 5,162,222), or thebaculovirus 39K delayed-early gene promoter (U.S. Pat. No. 5,155,037;U.S. Pat. No. 5,162,222).

The DNA sequence encoding the subtilase or subtilase variant of theinvention may also, if necessary, be operably connected to a suitableterminator.

The recombinant vector of the invention may further comprise a DNAsequence enabling the vector to replicate in the host cell in question.

The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, or a geneencoding resistance to e.g. antibiotics like ampicillin, kanamycin,chloramphenicol, erythromycin, tetracycline, spectinomycine, neomycin,hygromycin, methotrexate, or resistance to heavy metals, virus orherbicides, or which provides for prototrophy or auxotrophs. Examples ofbacterial selectable markers are the dal genes from Bacillus subtilis orBacillus licheniformis, resistance. A frequently used mammalian markeris the dihydrofolate reductase gene (DHFR). Suitable markers for yeasthost cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Aselectable marker for use in a filamentous fungal host cell may beselected from the group including, but not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),trpC (anthranilate synthase), and glufosinate resistance markers, aswell as equivalents from other species. Particularly, for use in anAspergillus cell are the amdS and pyrG markers of Aspergillus nidulansor Aspergillus oryzae and the bar marker of Streptomyces hygroscopicus.Furthermore, selection may be accomplished by cotransformation, e.g., asdescribed in WO 91/17243, where the selectable marker is on a separatevector.

To direct a subtilase or subtilase variant of the present invention intothe secretory pathway of the host cells, a secretory signal sequence(also known as a leader sequence, prepro sequence or pre sequence) maybe provided in the recombinant vector. The secretory signal sequence isjoined to the DNA sequence encoding the enzyme in the correct readingframe. Secretory signal sequences are commonly positioned 5′ to the DNAsequence encoding the enzyme. The secretory signal sequence may be thatnormally associated with the enzyme or may be from a gene encodinganother secreted protein.

The procedures used to ligate the DNA sequences coding for the presentenzyme, the promoter and optionally the terminator and/or secretorysignal sequence, respectively, or to assemble these sequences bysuitable PCR amplification schemes, and to insert them into suitablevectors containing the information necessary for replication orintegration, are well known to persons skilled in the art (cf., forinstance, Sambrook et al.).

More than one copy of a nucleic acid sequence encoding an enzyme of thepresent invention may be inserted into the host cell to amplifyexpression of the nucleic acid sequence. Stable amplification of thenucleic acid sequence can be obtained by integrating at least oneadditional copy of the sequence into the host cell genome using methodswell known in the art and selecting for transformants.

The nucleic acid constructs of the present invention may also compriseone or more nucleic acid sequences which encode one or more factors thatare advantageous in the expression of the polypeptide, e.g., anactivator (e.g., a trans-acting factor), a chaperone, and a processingprotease. Any factor that is functional in the host cell of choice maybe used in the present invention. The nucleic acids encoding one or moreof these factors are not necessarily in tandem with the nucleic acidsequence encoding the polypeptide.

Host Cells

The DNA sequence encoding the subtilases and/or subtilase variants ofthe present invention may be either homologous or heterologous to thehost cell into which it is introduced. If homologous to the host cell,i.e. produced by the host cell in nature, it will typically be operablyconnected to another promoter sequence or, if applicable, anothersecretory signal sequence and/or terminator sequence than in its naturalenvironment. The term “homologous” is intended to include a DNA sequenceencoding an enzyme native to the host organism in question. The term“heterologous” is intended to include a DNA sequence not expressed bythe host cell in nature. Thus, the DNA sequence may be from anotherorganism, or it may be a synthetic sequence.

The host cell into which the DNA construct or the recombinant vector ofthe invention is introduced may be any cell that is capable of producingthe present subtilases and/or subtilase variants, such as prokaryotes,e.g. bacteria or eukaryotes, such as fungal cells, e.g. yeasts orfilamentous fungi, insect cells, plant cells or mammalian cells.

Examples of bacterial host cells which, on cultivation, are capable ofproducing the subtilases or subtilase variants of the invention aregram-positive bacteria such as strains of Bacillus, e.g. strains of B.subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus,B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.lautus, B. megaterium or B. thuringiensis, or strains of Streptomyces,such as S. lividans or S. murinus, or gram-negative bacteria such asEscherichia coli or Pseudomonas sp.

The transformation of the bacteria may be effected by protoplasttransformation, electroporation, conjugation, or by using competentcells in a manner known per se (cf. Sambrook et al., supra).

When expressing the subtilases and/or subtilase variant in bacteria suchas E. coli, the enzyme may be retained in the cytoplasm, typically asinsoluble granules (known as inclusion bodies), or it may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed and the granules are recovered and denaturedafter which the enzyme is refolded by diluting the denaturing agent. Inthe latter case, the enzyme may be recovered from the periplasmic spaceby disrupting the cells, e.g. by sonication or osmotic shock, to releasethe contents of the periplasmic space and recovering the enzyme.

When expressing the subtilases and/or subtilase variant in gram-positivebacteria such as Bacillus or Streptomyces strains, the enzyme may beretained in the cytoplasm, or it may be directed to the extracellularmedium by a bacterial secretion sequence. In the latter case, the enzymemay be recovered from the medium as described below.

Examples of host yeast cells include cells of a species of Candida,Kluyveromyces, Saccharomyces, Schizosaccharomyces, Candida, Pichia,Hansenula, or Yarrowia. In a particular embodiment, the yeast host cellis a Saccharomyces carlsbergensis, Saccharomyces cerevisiae,Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyceskluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell. Otheruseful yeast host cells are a Kluyveromyces lactis Kluyveromycesfragilis Hansenula polymorpha, Pichia pastoris Yarrowia lipolytica,Schizosaccharomyces pombe, Ustilgo maylis, Candida maltose, Pichiaguillermondii and Pichia methanolio cell (cf. Gleeson et al., J. Gen.Microbiol. 132, 1986, pp. 3459-3465; U.S. Pat. No. 4,882,279 and U.S.Pat. No. 4,879,231). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980. The biology of yeast and manipulation of yeast genetics arewell known in the art (see, e.g., Biochemistry and Genetics of Yeast,Bacil, M., Horecker, B. J., and Stopani, A. O. M., editors, 2nd edition,1987; The Yeasts, Rose, A. H., and Harrison, J. S., editors, 2ndedition, 1987; and The Molecular Biology of the Yeast Saccharomyces,Strathern et al., editors, 1981). Yeast may be transformed using theprocedures described by Becker and Guarente, In Abelson, J. N. andSimon, M. I., editors, Guide to Yeast Genetics and Molecular Biology,Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., NewYork; Ito et al., 1983, Journal of Bacteriology 153:163; and Hinnen etal., 1978, Proceedings of the National Academy of Sciences USA 75:1920.

Examples of filamentous fungal cells include filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra), in particular it may of the a cell of a species ofAcremonium, such as A. chrysogenum, Aspergillus, such as A. awamori, A.foetidus, A. japonicus, A. niger, A. nidulans or A. oryzae, Fusarium,such as F. bactridioides, F. cerealis, F. crookwellense, F. culmorum, F.graminearum, F. graminum, F. heterosporum, F. negundi, F. reticulatum,F. roseum, F. sambucinum, F. sarcochroum, F. sulphureum, F.trichothecioides or F. oxysporum, Humicola, such as H. insolens or H.lanuginose, Mucor, such as M. miehei, Myceliophthora, such as M.thermophilum, Neurospora, such as N. crassa, Penicillium, such as P.purpurogenum, Thielavia, such as T. terrestris, Tolypocladium, orTrichoderma, such as T. harzianum, T. koningii, T. longibrachiatum, T.reesei or T. viride, or a teleomorph or synonym thereof. The use ofAspergillus spp. for the expression of proteins is described in, e.g.,EP 272 277, EP 230 023.

Examples of insect cells include a Lepidoptera cell line, such asSpodoptera frugiperda cells or Trichoplusia ni cells (cf. U.S. Pat. No.5,077,214). Culture conditions may suitably be as described in WO89/01029 or WO 89/01028. Transformation of insect cells and productionof heterologous polypeptides therein may be performed as described inU.S. Pat. No. 4,745,051; U.S. Pat. No. 4,775,624; U.S. Pat. No.4,879,236; U.S. Pat. No. 5,155,037; U.S. Pat. No. 5,162,222; EP397,485).

Examples of mammalian cells include Chinese hamster ovary (CHO) cells,HeLa cells, baby hamster kidney (BHK) cells, COS cells, or any number ofother immortalized cell lines available, e.g., from the American TypeCulture Collection. Methods of transfecting mammalian cells andexpressing DNA sequences introduced in the cells are described in e.g.Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601-621; Southern and Berg,J. Mol. Appl. Genet. 1 (1982), 327-341; Loyter et al., Proc. Natl. Acad.Sci. USA 79 (1982), 422-426; Wigler et al., Cell 14 (1978), 725; Corsaroand Pearson, Somatic Cell Genetics 7 (1981), 603, Ausubel et al.,Current Protocols in Molecular Biology, John Wiley and Sons, Inc., N.Y.,1987, Hawley-Nelson et al., Focus 15 (1993), 73; Ciccarone et al., Focus15 (1993), 80; Graham and van der Eb, Virology 52 (1973), 456; andNeumann et al., EMBO J. 1 (1982), 841-845. Mammalian cells may betransfected by direct uptake using the calcium phosphate precipitationmethod of Graham and Van der Eb (1978, Virology 52:546).

Methods for Expression and Isolation of Proteins

To express an enzyme of the present invention the above mentioned hostcells transformed or transfected with a vector comprising a nucleic acidsequence encoding an enzyme of the present invention are typicallycultured in a suitable nutrient medium under conditions permitting theproduction of the desired molecules, after which these are recoveredfrom the cells, or the culture broth.

The medium used to culture the host cells may be any conventional mediumsuitable for growing the host cells, such as minimal or complex mediacontaining appropriate supplements. Suitable media are available fromcommercial suppliers or may be prepared according to published recipes(e.g. in catalogues of the American Type Culture Collection). The mediamay be prepared using procedures known in the art (see, e.g., referencesfor bacteria and yeast; Bennett, J. W. and LaSure, L., editors, MoreGene Manipulations in Fungi, Academic Press, CA, 1991).

If the enzymes of the present invention are secreted into the nutrientmedium, they may be recovered directly from the medium. If they are notsecreted, they may be recovered from cell lysates. The enzymes of thepresent invention may be recovered from the culture medium byconventional procedures including separating the host cells from themedium by centrifugation or filtration, precipitating the proteinaceouscomponents of the supernatant or filtrate by means of a salt, e.g.ammonium sulfate, purification by a variety of chromatographicprocedures, e.g. ion exchange chromatography, gel filtrationchromatography, affinity chromatography, or the like, dependent on theenzyme in question.

The enzymes of the invention may be detected using methods known in theart that are specific for these proteins. These detection methodsinclude use of specific antibodies, formation of a product, ordisappearance of a substrate. For example, an enzyme assay may be usedto determine the activity of the molecule. Procedures for determiningvarious kinds of activity are known in the art.

The enzymes of the present invention may be purified by a variety ofprocedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing (IEF), differential solubility (e.g.,ammonium sulfate precipitation), or extraction (see, e.g., ProteinPurification, J-C Janson and Lars Ryden, editors, VCH Publishers, NewYork, 1989).

When an expression vector comprising a DNA sequence encoding an enzymeof the present invention is transformed/transfected into a heterologoushost cell it is possible to enable heterologous recombinant productionof the enzyme. An advantage of using a heterologous host cell is that itis possible to make a highly purified enzyme composition, characterizedin being free from homologous impurities, which are often present when aprotein or peptide is expressed in a homologous host cell. In thiscontext homologous impurities mean any impurity (e.g. other polypeptidesthan the enzyme of the invention) which originates from the homologouscell where the enzyme of the invention is originally obtained from.

Commercial Enzyme Applications

The present invention also relates to compositions comprising subtilaseand/or subtilase variants of the present invention. For example thesubtilase/subtilase variant may be used in compositions for personalcare, such as shampoo, soap bars, skin lotion, skin cream, hair dye,toothpaste, contact lenses, cosmetics, toiletries, or in compositionsused for treating textiles, for manufacturing food, e.g. baking or feed,or in compositions for cleaning purposes, e.g. detergents, dishwashingcompositions or for cleaning hard surfaces.

Detergents

The subtilase and/or subtilase variant of the invention may for examplebe used in detergent composition. It may be included in the detergentcomposition in the form of a non-dusting granulate, a stabilized liquid,or a protected enzyme. Non-dusting granulates may be produced, e.g., asdisclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionallybe coated by methods known in the art. Examples of waxy coatingmaterials are poly(ethylene oxide) products (polyethylene glycol, PEG)with mean molecular weights of 1000 to 20000; ethoxylated nonylphenolshaving from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols inwhich the alcohol contains from 12 to 20 carbon atoms and in which thereare 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; andmono- and di- and triglycerides of fatty acids. Examples of film-formingcoating materials suitable for application by fluid bed techniques aregiven in patent GB 1483591. Liquid subtilase/subtilase variantpreparations may, for instance, be stabilized by adding a polyol such aspropylene glycol, a sugar or sugar alcohol, lactic acid or boric acidaccording to established methods. Other enzyme stabilizers are wellknown in the art. Protected subtilase/subtilase variants may be preparedaccording to the method disclosed in EP 238,216.

The detergent composition may be in any convenient form, e.g. as powder,granules, paste or liquid. A liquid detergent may be aqueous, typicallycontaining up to 70% water and 0-30% organic solvent, or non-aqueous.

The detergent composition may comprise one or more surfactants, each ofwhich may be anionic, nonionic, cationic, or zwitterionic. The detergentwill usually contain 0-50% of anionic surfactant such as linearalkylbenzenesulfonate (LAS), alpha-olefinsulfonate (AOS), alkyl sulfate(fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or AES),secondary alkanesulfonates (SAS), alpha-sulfo fatty acid methyl esters,alkyl- or alkenylsuccinic acid, or soap. It may also contain 0-40% ofnonionic surfactant such as alcohol ethoxylate (AEO or AE), carboxylatedalcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside,alkyldimethylamine oxide, ethoxylated fatty acid monoethanolamide, fattyacid monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. asdescribed in WO 92/06154).

The detergent composition may additionally comprise one or more otherenzymes, such as e.g. proteases, amylases, lipolytic enzymes, cutinases,cellulases, peroxidases, oxidases, and further anti-microbialpolypeptides.

The detergent may contain 1-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate, citrate,nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTMPA), alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).The detergent may also be unbuilt, i.e. essentially free of detergentbuilder.

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP),polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA), polycarboxylatessuch as polyacrylates, maleic/acrylic acid copolymers and laurylmethacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine(TAED) or nonanoyloxybenzenesulfonate (NOBS). Alternatively, thebleaching system may comprise peroxyacids of, e.g., the amide, imide, orsulfone type.

The detergent composition may be stabilized using conventionalstabilizing agents, e.g. a polyol such as propylene glycol or glycerol,a sugar or sugar alcohol, lactic acid, boric acid, or a boric acidderivative such as, e.g., an aromatic borate ester, and the compositionmay be formulated as described in, e.g., WO 92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as, e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents,anti-soil-redeposition agents, dyes, bactericides, optical brighteners,or perfume.

The pH (measured in aqueous solution at use concentration) will usuallybe neutral or alkaline, e.g. in the range of 7-11.

Dishwashing Composition

Furthermore, the subtilases and/or subtilase variants of the presentinvention may also be used in dishwashing detergents.

Dishwashing detergent compositions typically comprise a surfactant whichmay be anionic, non-ionic, cationic, amphoteric or a mixture of thesetypes. The detergent may contain 0-90% of non-ionic surfactant such aslow- to non-foaming ethoxylated propoxylated straight-chain alcohols.

The detergent composition may contain detergent builder salts ofinorganic and/or organic types. The detergent builders may be subdividedinto phosphorus-containing and non-phosphorus-containing types. Thedetergent composition usually contains 1-90% of detergent builders.

Examples of phosphorus-containing inorganic alkaline detergent builders,when present, include the water-soluble salts especially alkali metalpyrophosphates, orthophosphates, and polyphosphates. An example ofphosphorus-containing organic alkaline detergent builder, when present,includes the water-soluble salts of phosphonates. Examples ofnon-phosphorus-containing inorganic builders, when present, includewater-soluble alkali metal carbonates, borates and silicates as well asthe various types of water-insoluble crystalline or amorphous aluminosilicates of which zeolites are the best-known representatives.

Examples of suitable organic builders include the alkali metal, ammoniumand substituted ammonium, citrates, succinates, malonates, fatty acidsulphonates, carboxymethoxy succinates, ammonium polyacetates,carboxylates, polycarboxylates, aminopolycarboxylates, polyacetylcarboxylates and polyhydroxsulphonates.

Other suitable organic builders include the higher molecular weightpolymers and copolymers known to have builder properties, for exampleappropriate polyacrylic acid, polymaleic and polyacrylic/polymaleic acidcopolymers and their salts.

The dishwashing detergent composition may contain bleaching agents ofthe chlorine/bromine-type or the oxygen-type. Examples of inorganicchlorine/bromine-type bleaches are lithium, sodium or calciumhypochlorite and hypobromite as well as chlorinated trisodium phosphate.Examples of organic chlorine/bromine-type bleaches are heterocyclicN-bromo and N-chloro imides such as trichloroisocyanuric,tribromoisocyanuric, dibromoisocyanuric and dichloroisocyanuric acids,and salts thereof with water-solubilizing cations such as potassium andsodium. Hydantoin compounds are also suitable.

The oxygen bleaches may be in the form of an inorganic persalt,particularly with a bleach precursor or as a peroxy acid compound.Examples of suitable peroxy bleach compounds include alkali metalperborates, e.g. tetrahydrates and monohydrates, alkali metalpercarbonates, persilicates and perphosphates. Particularly activatormaterials may be TAED and glycerol triacetate.

The dishwashing detergent composition may be stabilized usingconventional stabilizing agents for enzymes, e.g. a polyol such as e.g.propylene glycol, a sugar or a sugar alcohol, lactic acid, boric acid,or a boric acid derivative, e.g. an aromatic borate ester.

The dishwashing detergent composition may also contain otherconventional detergent ingredients, e.g. deflocculant material, fillermaterial, foam depressors, anti-corrosion agents, soil-suspendingagents, sequestering agents, anti-soil redeposition agents, dehydratingagents, dyes, bactericides, fluorescers, thickeners and perfumes.

Finally, the subtilases and/or subtilase variants of the invention maybe used in conventional dishwashing-detergents, e.g. in any of thedetergents described in any of the following patent publications:

-   EP 518719, EP 518720, EP 518721, EP 516553, EP 516554, EP 516555, GB    2200132, DE 3741617, DE 3727911, DE 4212166, DE 4137470, DE 3833047,    WO 93/17089, DE 4205071, WO 52/09680, WO 93/18129, WO 93/04153, WO    92/06157, WO 92/08777, EP 429124, WO 93/21299, U.S. Pat. No.    5,141,664, EP 561452, EP 561446, GB 2234980, WO 93/03129, EP 481547,    EP 530870, EP 533239, EP 554943, EP 346137, U.S. Pat. No. 5,112,518,    EP 318204, EP 318279, EP 271155, EP 271156, EP 346136, GB 2228945,    CA 2006687, WO 93/25651, EP 530635, EP 414197, U.S. Pat. No.    5,240,632.

Personal Care Applications

Another useful application area for the subtilases and/or subtilasevariants of the present invention is the personal care area where theend-user is in close contact with the protein, and where certainproblems with allergenicity has been encountered in experimental setups(Kelling et al., J. All. Clin. Imm., 1998, Vol. 101, pp. 179-187 andJohnston et al., Hum. Exp. Toxicol., 1999, Vol. 18, p. 527).

First of all the conjugate or compositions of the invention canadvantageously be used for personal care products, such as hair care andhair treatment products. This include products such as shampoo, balsam,hair conditioners, hair waving compositions, hair dyeing compositions,hair tonic, hair liquid, hair cream, hair rinse, hair spray.

Further contemplated are oral care products such as dentifrice, oralwashes, chewing gum.

Also contemplated are skin care products and cosmetics, such as skincream, skin milk, cleansing cream, cleansing lotion, cleansing milk,cold cream, cream soap, nourishing essence, skin lotion, milky lotion,calamine lotion, hand cream, powder soap, transparent soap, sun oil, sunscreen, shaving foam, shaving cream, baby oil lipstick, lip cream,creamy foundation, face powder, powder eye-shadow, powder, foundation,make-up base, essence powder, whitening powder.

Also for contact lenses hygiene products the subtilases and/or subtilasevariants of the invention may be used advantageously. Such productsinclude cleaning and disinfection products for contact lenses.

Food and Feed

The subtilase variants and/or subtilases of the present invention mayalso be used in food or feed products. For example said subtilasevariants/subtilases may be used modify the gluten phase of the dough,e.g. a hard wheat flour can be softened with a protease. Another exampleis within the brewery industry, where said subtilase variants/subtilasesmay be used for brewing with unmalted cereals and/or for controlling thenitrogen content.

Within the animal feed industry said subtilase variants and/orsubtilases may be used for so to speak expanding the animals' digestionsystem.

Materials and Methods

Materials

ELISA Reagents:

Horse Radish Peroxidase labelled pIg anti-rabbit-Ig (Dako, DK, P217,dilution 1:1000).

Mouse anti-rat IgE (Serotec MCA193; dilution 1:200).

Biotin-labelled mouse anti-rat IgG1 monoclonal antibody (Zymed 03-9140;dilution 1:1000)

Biotin-labelled rat anti-mouse IgG1 monoclonal antibody (SerotecMCA336B; dilution 1:2000)

Streptavidin-horse radish peroxidase (Kirkegård & Perry 14-30-00;dilution 1:1000).

OPD: o-phenylene-diamine, (Kementec cat no. 4260)

Rabbit anti-Savinase polyclonal IgG prepared by conventional means

Rat anti-Savinase polyclonal IgE prepared by conventional means.

Buffers and Solutions:

PBS (pH 7.2 (1 liter)) NaCl 8.00 g KCl 0.20 g K₂HPO₄ 1.04 g KH₂PO₄ 0.32g

Methods

Measurement of the Concentration of Specific IgE in the s.c. Mouse Modelby ELISA

The relative concentrations of specific IgE serum antibodies in the miceproduced in response to s.c. injection of proteins are measured by athree layer sandwich ELISA according to the following procedure:

-   -   1) The ELISA-plate was coated with 10 microgram rat anti-mouse        IgE (Serotech MCA419; dilution 1:100) Buffer 1 (50 microL/well).        Incubated over night at 4° C.    -   2) The plates were emptied and blocked with 2 (wt/v)% skim milk,        PBS for at least ½ hour at room temperature (200 microL/well).        Gently shaken. The plates were washed 3 times with 0.05 (v/v)%        Tween20, PBS.    -   3) The plates were incubated with mouse sera (50 microL/well),        starting from undiluted and continued with 2-fold dilutions.        Some wells were kept free for buffer 4 only (blanks). Incubated        for 30 minutes at room temperature. Gently shaken. The plates        were washed 3 times in 0.05 (v/v)% Tween20, PBS.    -   4) The subtilase or subtilase variant was diluted in 0.05 (v/v)%        Tween20, 0.5 (wt/v)% skim milk, PBS to the appropriate protein        concentration. 50 microL/well was incubated for 30 minutes at        room temperature. Gently shaken. The plates were washed 3 times        in 0.05 (v/v)% Tween20, PBS.    -   5) The specific polyclonal anti-subtilase or anti-subtilase        variant serum for detecting bound antibody was diluted in 0.05        (v/v)% Tween20, 0.5 (wt/v)% skim milk, PBS. 50 microl/well was        incubated for 30 minutes at room temperature. Gently shaken. The        plates were washed 3 times in 0.05 (v/v)% Tween20, PBS.    -   6) Horseradish Peroxidase-conjugated anti-pIg-antibody was        diluted in 0.05 (v/v)% Tween20, 0.5 (wt/v)% skim milk, PBS. 50        microL/well was incubated at room temperature for 30 minutes.        Gently shaken. The plates were washed 3 times in 0.05 (v/v)%        Tween20, PBS.    -   7) 0.6 mg ODP/ml+0.4 microL H₂O₂/ml were mixed in Citrate buffer        pH 5.2.    -   8) The solution was made just before use and incubated for 10        minutes.    -   9) 50 microL/well.    -   10) The reaction was stopped by adding 50 microL 2 N H₂SO₄/well.    -   11) The plates were read at 492 nm with 620 nm as reference.

Similar determination of IgG can be performed using anti mouse-IgG andstandard rat IgG reagents.

Measurement of the Concentration of Specific IgE in the MINT Assay byELISA

The relative concentrations of specific IgE serum antibodies in the miceproduced in response to intranasal dosing of proteins are measured by athree layer sandwich ELISA according to the following procedure:

-   -   1) The ELISA-plate (Nunc Maxisorp) was coated with 100        microliter/well rat anti-mouse IgE Heavy chain (HD-212-85-IgE3        diluted 1:100 in 0.05 M Carbonate buffer pH 9.6). Incubated over        night at 4° C.    -   2) The plates were emptied and blocked with 200 microliter/well        2% skim milk in 0.15 M PBS buffer pH 7.5 for 1 hour at 4° C. The        plates were washed 3 times with 0.15 M PBS buffer with 0.05%        Tween20.    -   3) The plates were incubated with dilutions of mouse sera (100        microL/well), starting from an 8-fold dilution and 2-fold        dilutions hereof in 0.15 M PBS buffer with 0.5% skim milk and        0.05% Tween20. Appropriate dilutions of positive and negative        control serum samples plus buffer controls were included.        Incubated for 1 hour at room temperature. Gently shaken. The        plates were washed 3 times in 0.15 M PBS buffer with 0.05%        Tween20.    -   4) 100 microliter/well of subtilase or subtilase variant diluted        to 1 microgram protein/ml in 0.15 M PBS buffer with 0.5% skim        milk and 0.05% Tween20 was added to the plates. The plates were        incubated for 1 hour at 4° C. The plates were washed 3 times        with 0.15 M PBS buffer with 0.05% Tween20.    -   5) The specific polyclonal anti-subtilase or anti-subtilase        variant serum for detecting bound antigen was diluted in 0.15 M        PBS buffer with 0.15% skim milk and 0.05% Tween20. 100        microl/well was incubated for 1 hour at 4° C. The plates were        washed 3 times in 0.15 M PBS buffer with 0.05% Tween20.    -   6) 100 microliter/well pig anti-rabbit Ig conjugated with        peroxidase diluted 1:1000 in 0.15 M PBS buffer with 0.5% skim        milk and 0.05% Tween20 was added to the plates. Incubated for 1        hour at 4° C. The plates were washed 3 times in 0.15 M PBS        buffer with 0.05% Tween20.    -   7) 250 microliter/well 0.1 M Citrat/phosphat buffer pH 5.0 was        added to the plates. Incubated for approximately 1 minute. The        plates were emptied.    -   8) 100 microliter/well ortho-phenylenediamine (OPD) solution (10        mg OPD diluted in 12.5 ml Citrat/phosphat buffer pH 5.0 and 12.5        microliter 30% hydrogen peroxide added just before use) was        added to the plates. Incubation for 4 minutes at room        temperature.    -   9) The reaction was stopped by adding 150 microliter/well 1M        H₂SO₄.    -   10) The plates were read at 490 nm with 620 nm as reference.

Examples Example 1

Identification of Epitopes in Savinase

A high diversity library of phages expressing random oligomeric peptides(hexa, hepta, octa, nona and/or dodeca peptides) as part of theirsurface proteins, were screened for their capacity to bind antibodies.The phage libraries were obtained from Schafer-N, Copenhagen, Denmarkand New England Biolabs Inc, UK.

Antibody samples were raised in animals (Rat, Rabbits or Mice) byparenteral or mucosal administration of each of the proteins listedbelow. The antibodies were dissolved in phosphate buffered saline (PBS).In some cases, antibodies of specific subclasses were purified from theserum of immunised animals by capryilic acid precipitation (for totalIgG) or by affinity chromatography using paramagnetic immunobeads (DynalAS) loaded with subtype-specific antibody (purification of IgG1, IgG4,IgE) or target protein, e.g. Bacillus lentus protease (Savinase™), forthe isolation of target specific Rat IgG, Mouse IgG, Mouse IgE, andRabbit IgG.

Protein-specific antibodies were also obtained from serum obtained fromsensitised humans.

The phage libraries were incubated with antibody coated beads. E.g.phages expressing oligopeptides with affinity for mouse IgE antibodiescaptured rat anti-mouse IgE-coated beads, and were collected by exposingthese paramagnetic beads to a magnetic field. The collected phages wereeluted from the immobilised antibodies by mild acid treatment, by directinfection of phage-sensitive E. coli strains, by elution with intactprotein antigen specific for the respective antibody sample (f.e.Savinase for anti-Savinase antibodies) or by a combination of theseapproaches. The isolated phages were amplified using methods known inthe art.

The specific phage-clones were isolated from the cell supernatant bycentrifugation in the presence of polyethylene glycol. DNA was isolated,the DNA sequence coding for the oligopeptide was amplified by PCR, andits DNA sequence was determined, all according to standard proceduresknown in the art. The amino acid sequence of the correspondingoligopeptide was deduced from the DNA sequence.

These experimentally determined reactive peptides were supplemented withinformation on reactive peptides published in the literature.

The sequences were collected in a database, and analysed by sequencealignment to identify epitope patterns.

Identifying Epitope Patterns

In principle, each of the reactive (oligo)peptide sequences representedan epitope pattern. However, some epitope patterns were redundant and toremove redundancy among the epitope patterns, the reactive(oligo)peptides sequences were subjected to computerised data analysis.

First all possible dipeptides were generated corresponding to 13²different combinations taking conservative alternatives into account.The presence and frequency of each dipeptide among the reactive(oligo)peptide sequences were listed. Next all possible tripeptides weregenerated corresponding to 13³ different combinations and again thepresence and frequency of each tripeptide among the reactive(oligo)peptide sequences were listed. All possible combinations of thelisted dipeptides and tripeptides were then generated including thosecontaining 1, 2, 3 or 4 residues inserted between the dipeptides andtripeptides, these residues selected among the possible residue types.This procedure generated a list of different peptide combinations ofamino acids each containing at least one dipeptide and at lest onetripeptide from the initial listings as well as 0 to 4 residues inbetween. The frequency of each peptide combination among the reactive(oligo)peptide sequences were then ranked and relevant epitope patternswere selected by a procedure where reactive peptides covered by the mostfrequent combination were first selected and separated from the group ofthe reactive peptides. Then reactive peptides covered by the second mostfrequent combination were selected and separated from the remaininggroup. Then reactive peptides covered by the third most frequentcombination were selected and separated from the remaining group. Thisprocedure was repeated until combinations covering all reactive peptidesare found.

Predicting Epitopes

The three-dimensional structure of SEQ ID NO:1 has been described inBetzel, C. et al.: “Crystal structure of the alkaline proteinaseSavinase from Bacillus lentus at 1.4 A resolution” in Journal ofMolecular Biology 223 pp. 427 (1992).

Surface accessibility was measured for each amino acid in SEQ ID NO:1using the DSSP program (see W. Kabsch and C. Sander, Biopolymers 22(1983) 2577-2637) in percent of a standard value for that amino acid.The standard values generated according to established methods byanalysing average surface accessibility of an amino acid in a 20-merhomogeneous peptide in helix formation using the DSSP program. For eachof the 13 different residue types (taking conservative alternatives intoconsideration) the average surface accessibility were as follows:

Accessibility Residue Å² A 62 C 92 D 69 E 156 F 123 G 50 H 130 I 84 K174 L 97 M 103 N 85 P 67 Q 127 R 211 S 64 T 80 V 81 W 126 Y 104

Epitopes were predicted by a computer program on a 3-dimensional modelof SEQ ID NO:1 by using the epitope patterns found as described above asfollows:

-   -   (1) For all amino acids in SEQ ID NO:1 it was examined if (a)        the amino acid type match the first amino acid of an epitope        patterns and (b) the solvent surface accessibility greater than        or equal to a predefined value, e.g., 20%. Those amino acid        satisfying 1(a) and 1(b) are selected.    -   (2) For all amino acids within a distance of 10 Å from the amino        acids selected in step 1 it is examined if (a) the amino acid        type matches the second amino acid of the pattern and (b) the        surface accessibility greater than or equal to the predefined        value, e.g., 20%. Those amino acid satisfying 2(a) and 2(b) are        selected    -   (3) For all amino acids within a distance 10 Å from the amino        acids selected in step 2 it is examined if (a) the amino acid        type matches the third amino acid of the pattern and (b) the        surface accessibility greater than or equal to the predefined        value, e.g., 20%. Those amino acid satisfying 3(a) and 3(b) are        selected.    -   (4) Repeating step 3 for all amino acids in the epitope pattern

Further, a limit of 25 Å was set as the maximum distance between any twoepitope residues.

This procedure was carried out for all epitope patterns for each of thefollowing settings for surface accessibility cut-off: 30, 40, 50, 60, 70and 80%. Epitope patterns finding a match on the 3 dimensional structureof SEQ ID NO: 1 according this procedure is predicted as epitopes.

Finally, for each of the seven settings for solvent accessibility, atable of all amino acids of SEQ ID NO:1 was created, in which each aminoacid residue was given a score by adding up the number of times itappeared in one of the epitopes (at that solvent setting). This scorewill be an indication of the likelihood that modification (substitution,insertion, deletion, glycosylation or chemical conjugation) of thatamino acid will, result in a variant of lower antigenicity. All aminoacids of the protein can then be ranked according to this score andthose with highest scores can be selected for mutagenesis.

By studying the positions of the top 10% scoring amino acids incombination with topological mapping as described below on thethree-dimensional model of SEQ ID NO:1, it is possible to identifyrelevant epitopes.

Topological Analysis of the Epitope Mapping Data

To get insight in the actual epitopes, coherent patches of amino acidswere identified by visualising the mapping data on the relevant3D-structure. Segments of ≧5 consecutive amino acids, the minimal lengthof each motif, were defined as the building blocks of the potentialepitopes. For each of the identified segments, the neighbouring aminoacids were determined using the SwissProt Pdb.viewer (www.ExPASy.com).Neighbouring was defined empirically as being situated within 8 Å of anyof the amino acids in the selected amino acid segment.

Applying the above described tools it has been possible to define 4epitopes on the molecule. These are:

Epitope 1: P52-N62; A98-S105; G127-E136; Y167; R170.

Epitope 2: G127; P129; N155-S161; Y167; R170; S188; Y192-G195; N218;N261-L262.

Epitope 3: A1-W6; P14-N18; T38-I44; Q59-N62; L75-I79; S87; N155;N204-N218.

Epitope 4: N6; Q12; G157-S161; D181-N185; Y192-G195; T255-S265.

Example 2

Library Construction

Applying the procedure outlined in Example 1 above, the subtilasevariants may be obtained by mutagenesis of the corresponding nucleicacid sequences as described in for example Sambrook et al. (1989),Molecular Cloning. A Laboratory Manual, Cold Spring Harbour, NY).

Example 3

Cloning and Expression of Subtilase Variants

Variants of the invention comprising specificinsertions/deletions/substitutions are made by traditional cloning ofDNA fragments (Sambrook et al., Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor, 1989) produced by PCR with oligoscontaining the desired mutations.

The template plasmid DNA may be pSX222, or an analogue of thiscontaining a variant of the enzyme in SEQ ID NO 1. Mutations areintroduced by oligo directed mutagenesis to the construction ofvariants.

The variants are transformed into E. coli. DNA purified from an overnight culture of these transformants is transformed into B. subtilis byrestriction endonuclease digestion, purification of DNA fragments,ligation, transformation of B. subtilis. Transformation of B. subtilisis performed as described by Dubnau et al., 1971, J. Mol. Biol. 56, pp.209-221.

Site-Directed Mutagenesis in Order to Introduce Mutations in a SpecificRegion:

The overall strategy used to perform site-directed mutagenesis is:

Mutagenic primers (oligonucleotides) are synthesized corresponding tothe DNA sequence flanking the sites of mutation, separated by the DNAbase pairs defining the insertions/deletions/substitutions.

Subsequently, the resulting mutagenic primers are used in a PCR reactionwith the modified plasmid pSX222. The resulting PCR fragment is purifiedand extended in a second PCR-reaction, the resulting PCR product ispurified and extended in a third PCR-reaction before being digested byendonucleases and cloned into the E. coli-B. subtilis shuttle vectorpSX222. The PCR reactions are performed under normal conditions. Theplasmid DNA is transformed into E. coli by well-known techniques and oneE. coli colony is sequenced to confirm the mutation designed.

Fermentation

Fermentations for the production of subtilase enzymes were performed at37° C. on a rotary shaking table (300 r.p.m.) in 500 ml baffledErlenmeyer flasks containing 100 ml BPX medium for 5 days.

Consequently, in order to make e.g. a 2 litre broth 20 Erlenmeyer flaskswere fermented simultaneously.

Media:

BPX Medium Composition (per litre) Potato starch 100 g Ground barley  50g Soybean flour  20 g Na2HPO4 × 12H2O  9 g Pluronic  0.1 g Sodiumcaseinate  10 g

The starch in the medium was liquefied with α-amylase and the medium wassterilized by heating at 120° C. for 45 minutes. After sterilization thepH of the medium was adjusted to 9 by addition of NaHCO3 to 0.1 M.

Example 4

Purification of Subtilase Variants

This procedure relates to purification of 2 litre scale fermentation forthe production of the subtilases of the invention in a Bacillus hostcell.

Approximately 1.6 litres of fermentation broth was centrifuged at 5000rpm for 35 minutes in 1 litre beakers. The supernatants were adjusted topH 6.5 using 10% acetic acid and filtered on Seitz Supra S100 filterplates.

The filtrates were concentrated to approximately 400 ml using an AmiconCH2A UF unit equipped with an Amicon S1Y10 UF cartridge. The UFconcentrate was centrifuged and filtered at room temperature prior toabsorption on a Bacitracin affinity column at pH 7. The subtilase waseluted from the Bacitracin column at room temperature using 25%2-propanol and 1 M sodium chloride in a buffer solution with 0.01dimethylglutaric acid, 0.1 M boric acid and 0.002 M calcium chlorideadjusted to pH 7.

The fractions with protease activity from the Bacitracin purificationstep were combined and applied to a 750 ml Sephadex G25 column (5 cmdiameter) equilibrated with a buffer containing 0.01 dimethylglutaricacid, 0.2 M boric acid and 0.002 m calcium chloride adjusted to pH 6.5.

By using the techniques mentioned above for the construction andfermentation, and the above isolation procedure, the novel subtilase ofthe present invention was produced and isolated.

This procedure was applied when purifying the subtilases of the presentinvention listed in table 1.

The procedure described in WO 03/037914 (Novozymes A/S) was applied whenpurifying the subtilases of the present invention listed in table 2.

Example 5

Competitive ELISA

Immunoplates (Nunc Maxisorb; Nunc) are coated overnight at 4° C. with asuitable dose, or dose-range, of wild type allergen. The plates are thenwashed thoroughly with Phosphate Buffered Saline (PBS) containing 0.05%Tween 20 (PBST), and remaining binding sites are blocked with PBScontaining 2% Skim Milk Powder (SMP). Dilute in a dose-range therecombinant competitor protease polypeptide variant in PBS containing0.5% SMP, 0.05% Tween 20 and mix with antigen specific rabbit polyclonalantibody diluted typically 2000-10,000 times in the same buffer. Add themixture to each well and incubated 2 h at 20° C. under gentle agitation.

Following a thorough wash with PBST, the allergen-IgG complexes aredetected, by serial incubation with an anti-rabbit antibody (DAKO), andgoat anti-rabbit Ig coupled to horse-radish peroxidase. The enzymaticactivity is measured by adding TMB Plus (Kem-En-Tec) as substrate, andstopping the reaction with an equal volume of 0.2 M H₂SO₄, and detectingcolour development by measuring optical density at 450 nm (OD450) in anELISA plate reader. If antigen specific rabbit polyclonal antibody bindsto the protease polypeptide variant in solution, it will reduce bindingto the plate-bound wild type polypeptide, thus reducing the OD450.

The above procedure was applied on large number of variants. Selectionof interesting protease variants by competitive ELISA was based upon:

-   -   1. Residual binding (%) of antibody after competition with        increasing amounts of variant as compared to the plate bound        protein of SEQ ID NO: 1 with the insertion *99aE, and    -   2. Shift of the IC50% (IC: Inhibiting concentration) to higher        or lower concentrations as reflecting differences in binding        affinity.

Variants were evaluated upon either purification or micro-purification.

Residual binding (%) is the ratio between OD450 measured at the highestcompetitor concentration and OD450 measured in the absence ofcompetitor. The residual binding with the parent protein as competitoris typically 0%. Variants typically move in terms of residual bindingtowards 100%.

Differences in binding affinity are expressed as an X-fold increase ordecrease of the amount of variant required obtaining a 50% inhibition ofantibody binding to the parent protein, as compared to the parentprotein itself. The inventors surprisingly observed that higher affinityof the variant for anti-parent protein specific antibody correlated aswell very well with a decrease in allergenic potential in animals.Differences in binding affinity can not be determined for variantsrevealing a >50% residual binding.

Competitive ELISA of Purified Protease Variants

TABLE 1 Evaluation of purified protease variants Variant Washperformance Residual Binding no. Mutations at 5 nM binding affinity  1PS57P, *99aE, R247Q >REF. 11% 7.5x  2P *99aE, A158V >REF. 0% 0.6x  3P*99aE, E136G >REF. n.d. n.d.  4P *99aE, E136K <REF. n.d. n.d.  5P I79T,*99aE, Q191E >REF. 13% 4x    6P *99aE, S141N, S156D >REF. 0% 2x    7PT58A, *99aE, S156N as REF. 0% 1.5x  8P *99aE, S141G, S156N, Q191E asREF. 0% 3x    9P S78P, *99aE, N185S, Q206L >REF. 19% 17x   10P *99aE,G195P, T260L >REF. 35% n.m 11P *99aE, G160S, G211S, T260N >REF. 0% 2.6x12P *99aE, G195D, G211N, T260L >REF. 66% n.m 13P *99aE, G160S, G195P,G211S, T260N >REF. 57% n.m 14P *99aE, A158N, S161D >REF. 4% 2.3x 15P*99aE, S259N, T260I >REF. 0% 1.2x 16P *99aE, S259N >REF. 0% 1.2x 17P*99aE, S259R as REF. 0% 1.0x 18P T22A, *99aE, G160N, T260L >REF. 0% 2.9x19P *99aE, G160D, G195P, G211P, T260N >REF. 21% 31x   20P *99aE, G160S,G211D, T260L >REF. 0% 1.5x Explanation of abbreviations used in table 1above: n.m.: not measurable; n.d.: not determined. “>REF.” indicatesthat wash performance is better than reference enzyme. “<REF.” indicatesthat wash performance is pourer than reference enzyme. “as REF.”indicates that wash performance is as reference enzyme. The referenceenzyme is the enzyme disclosed in SEQ ID NO 1 with the insertion *99aE.

All the above candidates have subsequently been evaluated with respectto wash performance according to the procedure described in Example 6.

Results of the wash performance evaluation are shown in table 1.

Competitive ELISA of Micro-Purified Protease Variants

A number of variants were micro-purified; upon micro-purificationresidual binding and binding affinity was evaluated.

TABLE 2 Evaluation of micro-purified protease variants Residual Bindingid no Mutations binding affinity 922.03 S9G, *99aE, A158N, G160D, S161D,N184S n.m n.m. 922.06 P52S, *99aE, A158D, S161G 0% 0.6x 923.06 *99aE,T260P 0% 1.7x 923.14 *99aE, T260I 0% 0.6x 923.16 *99aE, S259P 0% 0.4x924.16 *99aE, G160S, S163T, G195S, G211S, K237R, G258A, T260L n.m n.m.924.20 G23S, *99aE, G160N, G195D, V203G n.m n.m. 923.23 *99aE, G160P,G195P, V205A, G211P, T260N n.m n.m. 924.24 V95A, *99aE, G211N, T260L n.mn.m. 924.25 T66S, *99aE, G160N, G195N, G211P, T260I n.m n.m. 924.26*99aE, G160S, G195P, G211P, T260L n.m n.m. 924.29 L82P, *99aE, I107T,G160N, Q182R, G211D, K251R, T260I n.m n.m. 926.04 Y91H, *99aE 0% 0.3x926.14 Q12R, *99aE, S156H 0% 0.5x 926.29 *99aE, S156H, K251E n.m n.m.923.07 *99aE, T260N 0% 2.6x 923.10 *99aE, T260D 0% 1.6x 924.13 *99aE,G195N, G211P, T260I n.m n.m. 924.14 *99aE, G160S, A172S, N204T, G211P,T260N n.m n.m. 924.15 *99aE, G160S, G211P, T260L n.m n.m. 924.17 *99aE,M175I, G195S, T260L n.m n.m. 924.18 W6L, *99aE, G160S, G195N, S256N,T260L n.m n.m. 925.1 *99aE, Y192M, M222V, L262Q 0% 0.1x 925.2 *99aE,Y192W, L262Q 0% 0.1x 926.27 *99aE, S156H, Q191E 0% 1.1x 926.28 N77D,*99aE, S156N, Q191N 0% 1.2x 926.30 *99aE, S141G, S156D, M175I 0% 1.8x926.31 *99aE, Q191N 0% 0.9x 926.32 *99aE, S156D 0% 0.6x 926.33 *99aE,S156N, Q191N, Y263H 0% 0.8x n.m.: not measurable

Example 6

Test of the Wash Performance of Variants

Wash conditions: Temperature 30° C., wash time 14 min, water hardness 6°dH, detergent concentration 1.5 g/l with enzyme concentration 5, 10 and30 nM. As benchmarking enzyme the enzyme of SEQ ID NO 1 with theinsertion *99aE was used.

The detergents were commercial detergents which were inactivated bymaking a detergent solution and heat it for 5 min. at 85° C. in themicrowave oven.

pH was “as is” in the current detergent solution and was not adjusted.

Water hardness was adjusted by adding CaCl2*2H2O; MgCl₂*6H₂O; NaHCO₃(Ca²⁺:Mg²⁺:HCO₃ ⁻=2:1:6) to milli-Q water.

The test material was polyester/cotton swatches soiled withblood/milk/carbon black (EMPA 117, available from EMPA testmaterials,Mövenstrasse 12, CH-9015 St. Gallen).

After wash the reflectance (R) of the test material was measured at 460nm using a J&M Tidas MMS spectrophotometer. The measurements were doneaccording to the manufacturers' protocol.

-   -   R_(Variant): Reflectance of test material washed with variant    -   R_(Blank): Reflectance of test material washed with no enzyme    -   delta-Reflectance Rvariant−Rblank

The higher the delta-Reflectance the better is the wash performance. Thedelta-Reflectance was calculated for the dosage 5 nM enzyme.

Example 7

Epithelial Cell Assay

Reduced allergenicity was verified in vitro by stimulation of humanairlifted epithelial cells with enzyme variants. The presence of M-CSF,secreted into the culture medium in the well in response to enzymeexposure was measured by sandwich ELISA. The dose response curve of theM-CSF response to enzyme variants and reference protein was compared(see table 3). Variants with reduced allergenicity and adjuvancy werefound to require higher doses in order to elicit comparable cytokineresponses.

Reference protein for this study was the enzyme of SEQ ID NO 1 with a*99aE insertion.

FIG. 3 shows a response curve for the enzymes of SEQ ID NO1 and SEQ IDNO1 with a *99aE insertion. The figure clearly indicates that a proteinshowing reduced M-CSF production as compared to the reference enzyme ofthe present example will also show a reduced M-CSF production whencompared with the enzyme of SEQ ID NO 1.

Cell Culture Reagents

Epithelial Cells and Cell Growth Medium:

The human type-II alveolar cell line, A549 derived from human lungcarcinoma, was purchased from American Type Culture Collection (ATCC).A549 cells were cultured in Dulbecco's MEM/NUT mix F12 (DMEM/F12) mediumwith L-Glutamine, 15 MM HEPES, 10.000 IE Penicillin/10.000 μg/mlStreptomycin (GIBCO Invitrogen) supplemented with 0.5% Ultroser G(BioSepra). This medium is referred to as cell culture medium.

A549 cells were cultured in tissue culture flasks in cell culture medium(37° C., 5% CO2 and 100% humidity). Before cells were added, tissueculture flasks were coated with 2% Ultroser G at room temperature. After10 min. incubation, Ultroser G was removed and cells in cell culturemedium were plated into flasks and incubated for 7-10 days. Medium waschanged every second day. When cells reached 70-80% confluence inmonolayers, they were passaged into a new 2% Ultroser G-coated tissueculture flask. Cell culture medium was removed from the tissue cultureflask and the adherent A549 cells were rinsed with Hank's Balanced Salts(HBSS) w/o Calcium and Magnesium (GIBCO). HBSS was removed and 1×Trypsin-EDTA solution was added and incubated for 10 min. (or until thecells detach) at 37° C. Fresh cell culture medium was added, aspiratedand medium with detached cells were dispensed into either a new 2%Ultroser G-coated tissue flask or seeded on 1% Ultroser G-coated 0.4 μmPolycarbonate membranes (inserts) (Nunc) in an airlifted human cellstimulation assay. Cell viability was analysed by trypan blue stainingand was found to be between 75-95%. Cells were used between passages80-95.

Human Airlifted Epithelial Cell Cultures

25 mm polycarbonate tissue culture inserts were placed in wells on atissue culture plate. The polycarbonate tissue culture inserts werecoated with 1% Ultroser G for 10 min. at room temperature. After 10 min.incubation, Ultroser G was removed. 1.5 ml of 1.33×10⁵ cells/ml in cellculture medium was seeded on the polycarbonate tissue culture insert and1.5 ml cell culture medium was added in the well. Cells were culturedfor 10 days in the insert with fresh cell culture medium added everysecond day in the insert and the well. On day 7 the cells were airliftedand a liquid-air interface was created. This was done by removal ofmedium from the inserts and the well. Fresh cell culture medium was thenadded to the well and the airlifted cell cultures were grown for 3 daysmore. On day 10, fresh cell culture medium was added to the well and 150μl of cell culture medium with increasing concentrations of enzyme wasadded to the insert (on top of the cells). After 4 hours enzymeincubation, the cell culture medium in the well (under the cells) washarvested and stored at −20° C. until analysis with cytokine ELISA.After removal of cell culture medium, cell viability was measured usingAlamarBlue assay (SeroTec). The polycarbonate insert was cut of into thewell and 1 ml of fresh cell culture medium in addition to 125 μlAlamarBlue was added to each well and incubated for 4 hours at 37° C.with 5% CO2. AlarmarBlue reduction was then measured fluorometricallyand cell viability was calculated for each dose of each enzyme varianttested (% viability=(signal with enzymes/signal with mediumalone)*100%). Viability on 90-110% was found acceptable.

Cytokine ELISA

The presence of the cytokine M-CSF secreted into the cell culture mediumin response to enzyme exposure was measured using a sandwich ELISA.

The following data were obtained using the cell epithelial assay onselected variants:

TABLE 3 M-CSF secretion of human airlifted epithelial cell cultures inresponse to enzyme variant stimulation [μg/ml enzyme variant tostimulate 300 pg/ml M-CSF production] relative to [μg/ml referenceVariant enzyme^(a) to stimulate 300 pg/ml M-CSF production] SEQ ID NO 1with 1^(a) *99aE insertion  5P 2.2  6P 1.2 10P 2.5 11P 1.3 13P 1.2 18P1.1 19P 1.5 ^(a)M-CSF production in response to enzyme stimulation inseparated experiments was normalized with reference protein. Thereference protein in this Example is SEQ ID NO 1 with *99aE insertion.

The data disclosed in table 3 shows that the two variants 5P and 10Phave a reduced allergenicity profile as compared to the referenceenzyme.

Example 8

Testing of SEQ ID NO 1 Variants for Reduced Allergenicity in Vivo (MINTAssay).

Mouse intranasal (MINT) model (Robinson et al., Fund. Appl. Toxicol. 34,pp. 15-24, 1996).

Mice were dosed intranasally with the proteins on the first and thirdday of the experiment and from thereon on a weekly basis for a period of5 weeks. Blood samples were taken 15, 31 and 45 days after the start ofthe dosing. Serum was subsequently analysed for IgG1 (day 15) or IgE(day 31 and 45) levels.

The variants 5P (I79T+*99aE+Q191E) and 10P (*99aE+G195P+T260L) (bothinactive) were compared to SEQ ID NO 1 (in 0.9% NaCl).

The mean titres are shown in FIG. 1:

The IgG1 and IgE titres are expressed as the reciprocal of the highestdilution giving a positive ELISA reading converted to log 2. A readingis regarded as positive if higher than the OD-mean+2×standard deviationof the negative controls. There were 6 mice per dose level and theresults are expressed as group mean titres.

From FIG. 1 it can be concluded that the variants 5P (I79T+*99aE+Q191E)and 10P (*99aE+G195P+T260L) have considerably less potential foreliciting the production of antigen specific IgG1 and IgE antibody thanthose of the benchmark proteins, SEQ ID NO 1.

Example 9

Test of Performance of Subtilase Variants in Automatic Dishwashing (ADW)

The performance of the subtilase of the invention in full scale ADW istested in a household dish wash composition using standard conditions.The soil used is an egg/milk mixture coated on a steel plate. Further, aballast soil containing various foodstuffs is added.

Example

-   -   Detergent: Commercial or model detergent.    -   Detergent dosage 5.0 g/l    -   pH As is.    -   Water hardness: 3° dH to 21° dH    -   Temperature: 45° C. to 65° C.    -   Enzyme concentration: 10 nM to 230 nM, based on the total volume        of wash water in the machine.    -   Test method: Egg/milk or egg yolk soiling on steel plates as        described below.    -   Machine: Bosch or other commercially available.    -   Wash program:    -   Tap water is used; the following steps are applied:

Time Step (seconds) Temperature Main wash 1200¹⁾ 50° C.²⁾ Rinse  300¹⁾39° C.²⁾ Dry 1530 65° C. ¹⁾Heating of tap water takes place during theindicated time interval. ²⁾Final temperature upon heating of tap water.

Egg/Milk Soiling for Full Scale ADW Test

Materials:

220 ml full cream milk

15 eggs, medium size

Steel plates, diameter 18 cm

The dish wash composition is heated at 85° C. for 5 minutes in amicrowave oven in order to inactivate enzyme activity in thecomposition.

Soiling of Steel Plates:

220 ml full cream milk is mixed with 15 raw eggs in a Braun UK 20kitchen machine for 2 minutes. After sieving, stainless steel plates aresoiled in the mixture by immersion.

The plates are dried overnight at room temperature in an uprightposition. The dried plates are then heated at 120° C. for 45 minutes inorder to denature the proteins on the surface.

Egg Yolk Soiling for Full Scale ADW Test

Materials:

3 dL pasteurized egg yolk.

Steel plates, diameter 18 cm

The dish wash composition is heated at 85° C. for 5 minutes in amicrowave oven in order to inactivate enzyme activity in thecomposition.

Soiling of Steel Plates:

The steel plates are weighed on a balance giving 3 decimals.

Approx. 3 dL pasteurized egg yolk is mixed thoroughly and sieved througha kitchen screen.

The egg yolk broth is rolled onto the plates in a thin layer, e.g. usinga paint roller. This is done twice (without drying in between and withthe roller dipped in egg yolk also the second time).

The resulting layer of egg yolk should be around 1 g.

The plates are left to dry for minimum 4 hours at room temperature.

The soiled plates and the racks are then lowered into boilingdemineralised water for precisely 30 seconds.

The plates are left to dry for 30 minutes at room temperature.

After drying at room temperature the plates are dried in an oven at 80°C. for 30 minutes.

The plates are left to cool at room temperature for 30-60 minutes afterwhich they are weighed again.

Upon washing and drying at room temperature the plates are dried in theoven at 80° C. for 30 min.

Again after cooling at room temperature for 30-60 minutes the plates areweighed.

ADW Experiments

For each experiment, 10 soiled plates are washed in accordance withconditions listed above. In addition to the soiled plates, the machineis filled up with 10 porcelain plates, 4 glasses, 4 cups and 16 piecesof cutlery.

Furthermore, 50 g of ballast slurry is added to the machine. Thecomposition of the slurry is as follows:

3000 g are made, and the following components are weighed out:

Step Materials Dosage (g) 1 Margarine 189 Lard 189 Deep-Fry Oil 189Gravy Powder 51 2 Rapeseed Oil 948 Egg 474 3 Ketchup 189 Mustard 189 4Double Cream, 38% fat 282 Full-Cream Milk, 3.5% fat 189 5 Potato Flour66 Wheat Flour 18 Quark powder 18 Benzoic Acid 9

-   -   1. Margarine, lard and deep-fry oil are melted at low        temperature. Afterwards sieved gravy powder is added—under good        stirring—and is cooled down to 40° C.    -   2. Rapeseed oil and egg are mixed.    -   3. Ketchup and mustard are added into the oil/egg mass followed        by 5 minutes mixing.    -   4. The under 1) produced fat/gravy (cooled) is slowly added to        the mixture produced in 3) and mixed for further 5 minutes.    -   5. Double cream and full-cream milk are added the mixture and        mixed for 5 minutes.    -   6. The last flours and powders (step 5 in the table) is added.        The ballast slurry is mixed to a smooth mass.    -   7. The Ballast slurry is weighed out in portions of 50 g.

Measurements and Calculations for Egg/Milk

The light reflection values (R-values) are measured at six differentlocations on the plates using a Minolta Chroma Meter (Type: CR-300).Measurements are made on clean plates (R_(clean)), on soiled platesafter heating (R_(soiled)) and on plates after wash (R_(after wash)).

The removed protein film (% RPF) is calculated according to the belowformula:

% RPF=100%×(R _(after wash) −R _(soiled))/(R _(clean) −R _(soiled))

Measurements and Calculations for Egg Yolk

Performance data originate from gravimetric measurements of the steelplates as clean, soiled and washed. The performance is calculated as:

${\% \mspace{14mu} {Removed}\mspace{14mu} {Protein}\mspace{14mu} {Film}\mspace{14mu} \left( {\% \mspace{14mu} {RPF}} \right)} = \frac{\begin{pmatrix}{{Weight}_{soiled} -} \\{Weight}_{washed}\end{pmatrix} \times 100}{{Weight}_{soiled} - {Weight}_{clean}}$

Data Analysis

% RPF is fitted as a function of mg enzyme protein added.

The data are fitted by means of a four-parameter logistic model that canbe written as:

F(z)=Y ₀ +V _(max) *C ^(λ)/(k _(s) ^(λ) +C ^(λ))

Where F(z) is the response calculated from Y₀ as the intercept,Y₀+V_(max) being maximum response, C the enzyme dosage and k_(s) beingthe half-saturation value. λ is the steepness parameter that in aMichaelis-Menten model is equal 1, but here it is equal or differentfrom one as we are allowing S-shaped curves to be fitted.

Each curve fit is compared to the performance of the reference enzyme.

1-31. (canceled)
 32. A variant of the parent subtilase of SEQ. ID NO: 1,comprising two or more mutations wherein at least one of the mutationsis in the epitope regions 1 to 4 of the parent subtilase and wherein theimmunogenicity of the variant is less than the immunogenecity of theparent subtilase.
 33. The variant according to claim 32, wherein one ofthe mutations is an insertion of glutamic acid after position
 99. 34.The variant according to claim 32, wherein all the mutations are in theepitope regions 1 to
 4. 35. The variant according to claim 33, whereinall further mutations are in the epitope regions 1 to
 4. 36. The variantaccording to claim 32, comprising at least one mutation in epitoperegion 1 and at least one mutation in epitope region
 2. 37. The variantaccording to claim 33, comprising at least one mutation in epitoperegion 1 and at least one mutation in epitope region
 2. 38. The variantaccording to claim 32, comprising at least one mutation in epitoperegion 3 and at least one mutation in epitope region
 4. 39. The variantaccording to claim 33, comprising at least one mutation in epitoperegion 3 and at least one mutation in epitope region
 4. 40. The variantaccording to claim 32, comprising at least one mutation in epitoperegion 2 and at least one mutation in epitope region
 3. 41. The variantaccording to claim 33, comprising at least one mutation in epitoperegion 2 and at least one mutation in epitope region
 3. 42. The variantaccording to claim 32, comprising a mutation at one or more of thepositions selected from the group consisting of positions 22, 141, 191,and
 247. 43. The variant according to claim 33, comprising a mutation atone or more of the positions selected from the list consisting ofposition 22, 141, 191, and
 247. 44. The variant according to claim 43,wherein a) when a substitution occurs at position 22 the substitutingamino acid is alanine; b) when a substitution occurs at position 141 thesubstituting amino acid is asparagine or glycine; c) when a substitutionoccurs at position 191 the substituting amino acid is glutamic acid; d)when a substitution occurs at position 247 the substituting amino acidis glutamine.
 45. The variant according to claim 32, wherein when asubstitution occurs in epitope region 1 the substitution occurs at oneor more of the positions 57, 58, and 136, wherein a) when a substitutionoccurs at position 57 the substituting amino acid is proline; b) when asubstitution occurs at position 58 the substituting amino acid isalanine c) when a substitution occurs at position 136 the substitutingamino acid is glycine or lysine.
 46. The variant according to claim 32,wherein the variant is selected from the group consisting of:S57P+*99aE+R247Q, *99aE+A158V, *99aE+E136G, *99aE+E136K,I79T+*99aE+Q191E, *99aE+S141N+S156D, T58A+*99aE+S156N,S78P+*99aE+N185S+Q206L, *99aE+A158N+S161D, and *99aE+S259N, wherein thepositions for substitution are numbered according to the amino acidsequence of subtilisin BPN′ as set forth in SEQ ID NO: 5 herein.
 47. Thevariant according to claim 46, wherein the variant is selected from thegroup of variants consisting of: I79T+*99aE+Q191E and*99aE+S141N+S156N+Q191E.
 48. A composition comprising a variantaccording to claim 32 and a surfactant.
 49. A DNA sequence encoding avariant of claim
 32. 50. A vector comprising a DNA sequence of claim 49.51. A host cell comprising a vector of claim 50.